Red light-activated depletion of drug-refractory glioblastoma stem cells and chemosensitization of an acquired-resistant mesenchymal phenotype.

Glioblastoma stem cells (GSCs) are potent tumor initiators resistant to radiochemotherapy, and this subpopulation is hypothesized to re-populate the tumor milieu due to selection following conventional therapies. Here, we show that 5-aminolevulinic acid (ALA) treatment-a pro-fluorophore used for fluorescence-guided cancer surgery-leads to elevated levels of fluorophore conversion in patient-derived GSC cultures, and subsequent red light-activation induces apoptosis in both intrinsically temozolomide chemotherapy-sensitive and -resistant GSC phenotypes. Red light irradiation of ALA-treated cultures also exhibits the ability to target mesenchymal GSCs (Mes-GSCs) with induced temozolomide resistance. Furthermore, sub-lethal light doses restore Mes-GSC sensitivity to temozolomide, abrogating GSC-acquired chemoresistance. These results suggest that ALA is not only useful for fluorescence-guided glioblastoma tumor resection, but that it also facilitates a GSC drug-resistance agnostic, red light-activated modality to mop up the surgical margins and prime subsequent chemotherapy.

Tunable Macroscopic Alignment of Self-Assembling Peptide Nanofibers.

Progress in the design and synthesis of nanostructured self-assembling systems has facilitated the realization of numerous nanoscale geometries, including fibers, ribbons, and sheets. A key challenge has been achieving control across multiple length scales and creating macroscopic structures with nanoscale organization. Here, we present a facile extrusion-based fabrication method to produce anisotropic, nanofibrous hydrogels using self-assembling peptides. The application of shear force coinciding with ion-triggered gelation is used to kinetically trap supramolecular nanofibers into aligned, hierarchical macrostructures. Further, we demonstrate the ability to tune the nanostructure of macroscopic hydrogels through modulating phosphate buffer concentration during peptide self-assembly. In addition, increases in the nanostructural anisotropy of fabricated hydrogels are found to enhance their strength and stiffness under hydrated conditions. To demonstrate their utility as an extracellular matrix-mimetic biomaterial, aligned nanofibrous hydrogels are used to guide directional spreading of multiple cell types, but strikingly, increased matrix alignment is not always correlated with increased cellular alignment. Nanoscale observations reveal differences in cell-matrix interactions between variably aligned scaffolds and implicate the need for mechanical coupling for cells to understand nanofibrous alignment cues. In total, innovations in the supramolecular engineering of self-assembling peptides allow us to decouple nanostructure from macrostructure and generate a gradient of anisotropic nanofibrous hydrogels. We anticipate that control of architecture at multiple length scales will be critical for a variety of applications, including the bottom-up tissue engineering explored here.

Tunable Macroscopic Alignment of Self-Assembling Peptide Nanofibers.

Fibrous proteins that comprise the extracellular matrix (ECM) guide cellular growth and tissue organization. A lack of synthetic strategies able to generate aligned, ECM-mimetic biomaterials has hampered bottom-up tissue engineering of anisotropic tissues and led to a limited understanding of cell-matrix interactions. Here, we present a facile extrusion-based fabrication method to produce anisotropic, nanofibrous hydrogels using self-assembling peptides. The application of shear force coinciding with ion-triggered gelation is used to kinetically trap supramolecular nanofibers into aligned, hierarchical structures. We establish how modest changes in phosphate buffer concentration during peptide self-assembly can be used to tune their alignment and packing. In addition, increases in the nanostructural anisotropy of fabricated hydrogels are found to enhance their strength and stiffness under hydrated conditions. To demonstrate their utility as an ECM-mimetic biomaterial, aligned nanofibrous hydrogels are used to guide directional spreading of multiple cell types, but strikingly, increased matrix alignment is not always correlated with increased cellular alignment. Nanoscale observations reveal differences in cell-matrix interactions between variably aligned scaffolds and implicate the need for mechanical coupling for cells to understand nanofibrous alignment cues. In total, innovations in the supramolecular engineering of self-assembling peptides allow us to generate a gradient of anisotropic nanofibrous hydrogels, which are used to better understand directed cell growth.

COVID-19: From emerging variants to vaccination.

The vigorous spread of SARS-CoV-2 resulted in the rapid infection of millions of people worldwide and devastation of not only public healthcare, but also social, educational, and economic infrastructures. The evolution of SARS-CoV-2 over time is due to the mutations that occurred in the genome during each replication. These mutated forms of SARS-CoV-2, otherwise known as variants, were categorized as variants of interest (VOI) or variants of concern (VOC) based on the increased risk of transmissibility, disease severity, immune escape, decreased effectiveness of current social measures, and available vaccines and therapeutics. The swift development of COVID-19 vaccines has been a great success for biomedical research, and billions of vaccine doses, including boosters, have been administered worldwide. BNT162b2 vaccine (Pfizer-BioNTech), mRNA-1273 (Moderna), ChAdOx1 nCoV-19 (AstraZeneca), and Janssen (Johnson & Johnson) are the four major COVID-19 vaccines that received early regulatory authorization based on their efficacy. However, some SARS-CoV-2 variants resulted in higher resistance to available vaccines or treatments. It has been four years since the first reported infection of SARS-CoV-2, yet the Omicron variant and its subvariants are still infecting people worldwide. Despite this, COVID-19 vaccines are still expected to be effective at preventing severe disease, hospitalization, and death from COVID. In this review, we provide a comprehensive overview of the COVID-19 pandemic focused on evolution of VOC and vaccination strategies against them.

Orthogonal Self-Assembly of Amphiphilic Peptide Hydrogels and Liposomes Results in Composite Materials with Tunable Release Profiles.

Self-assembled nanomaterials are promising candidates for drug delivery by providing a higher degree of spatiotemporal control compared to free drugs. However, challenges such as burst release, inadequate targeting, and drug-nanomaterial incompatibility leave room for improvement. The combination of orthogonal self-assembling systems can result in more useful materials that improve upon these weaknesses. In this work, we investigate an orthogonal self-assembling system of nanofibrous MultiDomain Peptide (MDP) hydrogels encapsulating liposomes. Both positively charged and negatively charged MDPs were prepared and mixed with positively charged, negatively charged, or zwitterionic liposomes for a total of six composites. We demonstrate that, despite both systems being amphiphilic, they are able to mix while retaining their independent identities. We show that changing the charge of either liposomes or MDPs does not hinder the self-assembly of either system or significantly affect their rheological properties. In all six cases, small molecules encapsulated in liposome-MDP composites resulted in slower release than was possible in MDP hydrogels alone. However, in one case, positively charged MDPs destabilized negatively charged liposomes and resulted in a unique release profile. Finally, we show that MDP hydrogels substantially decrease the release of chemotherapeutic doxorubicin from its liposomal formulation, Doxil, for 24 h. This work demonstrates the chemical compatibility of amphiphilic, orthogonally self-assembled systems and the range of their drug-delivering capabilities.

Thermogelling hydrogel charge and lower critical solution temperature influence cellular infiltration and tissue integration in an ex vivo cartilage explant model.

Thermogelling hydrogels based on poly(N-isopropyl acrylamide) (p[NiPAAm]) and crosslinked with a peptide-bearing macromer poly(glycolic acid)-poly(ethylene glycol)-poly(glycolic acid)-di(but-2-yne-1,4-dithiol) (PdBT) were fabricated to assess the role of hydrogel charge and lower critical solution temperature (LCST) over time in influencing cellular infiltration and tissue integration in an ex vivo cartilage explant model over 21 days. The p(NiPAAm)-based thermogelling polymer was synthesized to possess 0, 5, and 10 mol% dimethyl-γ-butyrolactone acrylate (DBA) to raise the LCST over time as the lactone rings hydrolyzed. Further, three peptides were designed to impart charge into the hydrogels via conjugation to the PdBT crosslinker. The positively, neutrally, and negatively charged peptides K4 (+), zwitterionic K2E2 (0), and E4 (-), respectively, were conjugated to the modular PdBT crosslinker and the hydrogels were evaluated for their thermogelation behavior in vitro before injection into the cartilage explant models. Samples were collected at days 0 and 21, and tissue integration and cellular infiltration were assessed via mechanical pushout testing and histology. Negatively charged hydrogels whose LCST changed over time (10 mol% DBA) were demonstrated to promote the greatest tissue integration when compared to the positive and neutral gels of the same thermogelling polymer formulation due to increased transport and diffusion across the hydrogel-tissue interface. Indeed, the negatively charged thermogelling polymer groups containing 5 and 10 mol% DBA demonstrated cellular infiltration and cartilage-like matrix deposition via histology. This study demonstrates the important role that material physicochemical properties play in dictating cell and tissue behavior and can inform future cartilage tissue engineering strategies.

Remediating Desmoplasia with EGFR-Targeted Photoactivable Multi-Inhibitor Liposomes Doubles Overall Survival in Pancreatic Cancer.

Desmoplasia is characteristic of pancreatic ductal adenocarcinoma (PDAC), which exhibits 5-year survival rates of 3%. Desmoplasia presents physical and biochemical barriers that contribute to treatment resistance, yet depleting the stroma alone is unsuccessful and even detrimental to patient outcomes. This study is the first demonstration of targeted photoactivable multi-inhibitor liposomes (TPMILs) that induce both photodynamic and chemotherapeutic tumor insult, while simultaneously remediating desmoplasia in orthotopic PDAC. TPMILs targeted with cetuximab (anti-EGFR mAb) contain lipidated benzoporphyrin derivative (BPD-PC) photosensitizer and irinotecan. The desmoplastic tumors comprise human PDAC cells and patient-derived cancer-associated fibroblasts. Upon photoactivation, the TPMILs induce 90% tumor growth inhibition at only 8.1% of the patient equivalent dose of nanoliposomal irinotecan (nal-IRI). Without EGFR targeting, PMIL photoactivation is ineffective. TPMIL photoactivation is also sixfold more effective at inhibiting tumor growth than a cocktail of Visudyne-photodynamic therapy (PDT) and nal-IRI, and also doubles survival and extends progression-free survival by greater than fivefold. Second harmonic generation imaging reveals that TPMIL photoactivation reduces collagen density by >90% and increases collagen nonalignment by >103 -fold. Collagen nonalignment correlates with a reduction in tumor burden and survival. This single-construct phototoxic, chemotherapeutic, and desmoplasia-remediating regimen offers unprecedented opportunities to substantially extend survival in patients with otherwise dismal prognoses.

A Combined Conduit-Bioactive Hydrogel Approach for Regeneration of Transected Sciatic Nerves.

Transected peripheral nerve injury (PNI) affects the quality of life of patients, which leads to socioeconomic burden. Despite the existence of autografts and commercially available nerve guidance conduits (NGCs), the complexity of peripheral nerve regeneration requires further research in bioengineered NGCs to improve surgical outcomes. In this work, we introduce multidomain peptide (MDP) hydrogels, as intraluminal fillers, into electrospun poly(ε-caprolactone) (PCL) conduits to bridge 10 mm rat sciatic nerve defects. The efficacy of treatment groups was evaluated by electromyography and gait analysis to determine their electrical and motor recovery. We then studied the samples' histomorphometry with immunofluorescence staining and automatic axon counting/measurement software. Comparison with negative control group shows that PCL conduits filled with an anionic MDP may improve functional recovery 16 weeks postoperation, displaying higher amplitude of compound muscle action potential, greater gastrocnemius muscle weight retention, and earlier occurrence of flexion contracture. In contrast, PCL conduits filled with a cationic MDP showed the least degree of myelination and poor functional recovery. This phenomenon may be attributed to MDPs' difference in degradation time. Electrospun PCL conduits filled with an anionic MDP may become an attractive tissue engineering strategy for treating transected PNI when supplemented with other bioactive modifications.

Evaluating the physicochemical effects of conjugating peptides into thermogelling hydrogels for regenerative biomaterials applications.

Thermogelling hydrogels, such as poly(N-isopropylacrylamide) [P(NiPAAm)], provide tunable constructs leveraged in many regenerative biomaterial applications. Recently, our lab developed the crosslinker poly(glycolic acid)-poly(ethylene glycol)-poly(glycolic acid)-di(but-2-yne-1,4-dithiol), which crosslinks P(NiPAAm-co-glycidyl methacrylate) via thiol-epoxy reaction and can be functionalized with azide-terminated peptides via alkyne-azide click chemistry. This study's aim was to evaluate the impact of peptides on the physicochemical properties of the hydrogels. The physicochemical properties of the hydrogels including the lower critical solution temperature, crosslinking times, swelling, degradation, peptide release and cytocompatibility were evaluated. The gels bearing peptides increased equilibrium swelling indicating hydrophilicity of the hydrogel components. Comparable sol fractions were found for all groups, indicating that inclusion of peptides does not impact crosslinking. Moreover, the inclusion of a matrix metalloproteinase-sensitive peptide allowed elucidation of whether release of peptides from the network was driven by hydrolysis or enzymatic cleavage. The hydrophilicity of the network determined by the swelling behavior was demonstrated to be the most important factor in dictating hydrogel behavior over time. This study demonstrates the importance of characterizing the impact of additives on the physicochemical properties of hydrogels. These characteristics are key in determining design considerations for future in vitro and in vivo studies for tissue regeneration.

One-Step Detection and Classification of Bacterial Carbapenemases in 10 Minutes Using Fluorescence Identification of ß-Lactamase Activity.

Rapid and accurate diagnosis of bacterial carbapenemases remains a major challenge for clinical laboratories. A novel assay was developed here using fluorescence identification of ß-lactamase activity (FIBA) to permit rapid detection and classification of bacterial carbapenemases. By mixing a fluorogenic ß-lactamase substrate, ß-LEAF (ß-lactamase enzyme-activated fluorophore), with bacterial isolates plus the respective inhibitor (imipenem for noncarbapenemase ß-lactamases, clavulanic acid for type A carbapenemases, and EDTA for type B carbapenemases), objective results with 95% to 100% sensitivity and specificity were generated in 10 min. FIBA is ≈$1/test and consists of only a single mixing step. Given the combination of rapidity, accuracy, low cost, and simplicity, this novel carbapenemase detection and classification assay is well positioned to be applied in clinical microbiology laboratories to provide guidance for the choice of proper treatment and control of globally prevalent carbapenemase-positive infections.

NIR Photodynamic Destruction of PDAC and HNSCC Nodules Using Triple-Receptor-Targeted Photoimmuno-Nanoconjugates: Targeting Heterogeneity in Cancer.

Receptor heterogeneity in cancer is a major limitation of molecular targeting for cancer therapeutics. Single-receptor-targeted treatment exerts selection pressures that result in treatment escape for low-receptor-expressing tumor subpopulations. To overcome this potential for heterogeneity-driven resistance to molecular targeted photodynamic therapy (PDT), we present for the first time a triple-receptor-targeted photoimmuno-nanoconjugate (TR-PIN) platform. TR-PIN functionalization with cetuximab, holo-transferrin, and trastuzumab conferred specificity for epidermal growth factor receptor (EGFR), transferrin receptor (TfR), and human epidermal growth factor receptor 2 (HER-2), respectively. The TR-PINs exhibited up to a 24-fold improvement in cancer cell binding compared with EGFR-specific cetuximab-targeted PINs (Cet-PINs) in low-EGFR-expressing cell lines. Photodestruction using TR-PINs was significantly higher than the monotargeted Cet-PINs in heterocellular 3D in vitro models of heterogeneous pancreatic ductal adenocarcinoma (PDAC; MIA PaCa-2 cells) and heterogeneous head and neck squamous cell carcinoma (HNSCC, SCC9 cells) containing low-EGFR-expressing T47D (high TfR) or SKOV-3 (high HER-2) cells. Through their capacity for multiple tumor target recognition, TR-PINs can serve as a unique and amenable platform for the effective photodynamic eradication of diverse tumor subpopulations in heterogeneous cancers to mitigate escape for more complete and durable treatment responses.

Photoimmunotherapy of Ovarian Cancer: A Unique Niche in the Management of Advanced Disease.

Ovarian cancer (OvCa) is the leading cause of gynecological cancer-related deaths in the United States, with five-year survival rates of 15-20% for stage III cancers and 5% for stage IV cancers. The standard of care for advanced OvCa involves surgical debulking of disseminated disease in the peritoneum followed by chemotherapy. Despite advances in treatment efficacy, the prognosis for advanced stage OvCa patients remains poor and the emergence of chemoresistant disease localized to the peritoneum is the primary cause of death. Therefore, a complementary modality that is agnostic to typical chemo- and radio-resistance mechanisms is urgently needed. Photodynamic therapy (PDT), a photochemistry-based process, is an ideal complement to standard treatments for residual disease. The confinement of the disease in the peritoneal cavity makes it amenable for regionally localized treatment with PDT. PDT involves photochemical generation of cytotoxic reactive molecular species (RMS) by non-toxic photosensitizers (PSs) following exposure to non-harmful visible light, leading to localized cell death. However, due to the complex topology of sensitive organs in the peritoneum, diffuse intra-abdominal PDT induces dose-limiting toxicities due to non-selective accumulation of PSs in both healthy and diseased tissue. In an effort to achieve selective damage to tumorous nodules, targeted PS formulations have shown promise to make PDT a feasible treatment modality in this setting. This targeted strategy involves chemical conjugation of PSs to antibodies, referred to as photoimmunoconjugates (PICs), to target OvCa specific molecular markers leading to enhanced therapeutic outcomes while reducing off-target toxicity. In light of promising results of pilot clinical studies and recent preclinical advances, this review provides the rationale and methodologies for PIC-based PDT, or photo-immunotherapy (PIT), in the context of OvCa management.

Analysis of radiation-induced changes to human melanoma cultures using a mathematical model.

OBJECTIVES: Tumour cells respond to ionizing radiation by cycle arrest, cell death or repair and possible regrowth. We have developed a dynamic mathematical model of the cell cycle to incorporate transition probabilities for entry into DNA replication and mitosis. In this study, we used the model to analyse effects of radiation on cultures of five human melanoma cell lines. MATERIALS AND METHODS: Cell lines were irradiated (9 Gy) prior to further culture and harvesting at multiple points up to 96 h later. Cells were fixed, stained with propidium iodide and analysed for G(1)-, S- and G(2)/M-phase cells by flow cytometry. Data for all time points were fitted to a mathematical model. To provide unique solutions, cultures were grown in the presence and absence of the mitotic poison paclitaxel, added to prevent cell division. RESULTS: The model demonstrated that irradiation at 9 Gy induced G(2)-phase arrest in all lines for at least 96 h. Two cell lines with wild-type p53 status additionally exhibited G(1)-phase arrest with recovery over 15 h, as well as evidence of cell loss. Resumption of cycling of surviving cells, as indicated by increases in G(1)/S and G(2)/M-phase transitions, was broadly comparable with results of clonogenic assays. CONCLUSIONS: The results, combined with existing data from clonogenic survival assays, support the hypothesis that a dominant effect of radiation in these melanoma lines is the induction of long-term cell cycle arrest.

Induction of intratumoral tumor necrosis factor (TNF) synthesis and hemorrhagic necrosis by 5,6-dimethylxanthenone-4-acetic acid (DMXAA) in TNF knockout mice.

5,6-Dimethylxanthenon-4-acetic acid (DMXAA) is a new antitumor drug currently undergoing clinical trial. Administration of DMXAA to mice with tumors leads to cessation of tumor blood flow and the onset of tumor hemorrhagic necrosis, accompanied by the production of the cytokine tumor necrosis factor (TNF). Previous studies have shown that DMXAA induces both tumor and host cells to synthesize TNF and that induced intratumoral TNF production correlates with the antitumor activity of DMXAA. To explore the hypothesis that TNF production by tumor cells contributed to the induction of hemorrhagic necrosis by DMXAA, TNF-/- (C57Bl/6 background) mice were used as recipients for the s.c. implantation of (TNF positive) colon 38 adenocarcinoma. Tumors removed 24 h after treatment with DMXAA (66 or 100 micromol/kg) were found to be hemorrhagic and necrotic. Cells expressing TNF mRNA in tumors removed 2 h after treatment with DMXAA (160 micromol/kg) were found by in situ hybridization to be comparable in frequency and distribution with those in tumors from C57Bl/6 TNF-positive mice. However, the amount of TNF protein extracted from tumors from TNF knockout mice was lower than that from TNF-positive mice. Spleen and liver tissue from TNF knockout mice, in contrast to that from TNF-positive mice, produced no TNF mRNA. TNF protein was undetectable in liver and spleen tissue from TNF knockout mice, but was evident in tissue from TNF-positive mice. These results confirm that DMXAA has the novel ability of inducing tumors to synthesize TNF in situ.

Thalidomide increases both intra-tumoural tumour necrosis factor-alpha production and anti-tumour activity in response to 5,6-dimethylxanthenone-4-acetic acid.

5,6-Dimethylxanthenone-4-acetic acid (DMXAA), synthesized in this laboratory and currently in phase I clinical trial, is a low molecular weight inducer of tumour necrosis factor-alpha (TNF-alpha). Administration of DMXAA to mice with established transplantable tumours elicits rapid vascular collapse selectively in the tumour, followed by extensive haemorrhagic necrosis mediated primarily through the production of TNF-alpha. In this report we have investigated the synthesis of TNF-alpha mRNA in hepatic, splenic and tumour tissue. Co-administration of thalidomide with DMXAA increased anti-tumour activity and increased intra-tumoural TNF-alpha production approximately tenfold over that obtained with DMXAA alone. Thalidomide increased splenic TNF-alpha production slightly but significantly decreased serum and hepatic levels of TNF-alpha induced with DMXAA. Lipopolysaccharide (LPS) induced 300-fold higher serum TNF-alpha than did DMXAA at the maximum tolerated dose, but induced similar amounts of TNF-alpha in spleen, liver and tumour. Splenic TNF-alpha activity induced with LPS was slightly increased with thalidomide, but serum and liver TNF-alpha levels were suppressed. Thalidomide did not increase intra-tumoural TNF-alpha production induced with LPS, in sharp contrast to that obtained with DMXAA. While thalidomide improved the anti-tumour response to DMXAA, it had no effect on the anti-tumour action of LPS that did not induce a significant growth delay or cures against the Colon 38 tumour. The increase in the anti-tumour action by thalidomide in combination with DMXAA corresponded to an increase in intra-tumoural TNF-alpha production. Co-administration of thalidomide may represent a novel approach to improving selective intra-tumoural TNF-alpha production and anti-tumour efficacy of DMXAA.

Stimulation of tumors to synthesize tumor necrosis factor-alpha in situ using 5,6-dimethylxanthenone-4-acetic acid: a novel approach to cancer therapy.

The selective induction of tumor vascular collapse represents an exciting approach to cancer treatment. However, clinical evaluation of tumor necrosis factor-alpha (TNF), an agent that accomplishes this goal, has been limited by systemic toxicity, and clinical approaches using bacterial components to induce TNF production have also been disappointing. Our laboratory has developed synthetic low molecular weight inducers of TNF, including 5,6-dimethylxanthenone-4-acetic acid (DMXAA), as an alternative strategy. DMXAA induces rapid vascular collapse in transplantable murine tumors and induces TNF synthesis in vitro in both murine and human systems. We show here that the extent of DMXAA-induced TNF synthesis is greater in tumors than that in the spleen, liver, or serum. As shown by in situ hybridization studies of the murine Colon 38 tumor, DMXAA induced tumor as well as host cells to express TNF mRNA. The distribution of cells containing TNF mRNA in tumor tissues after DMXAA administration contrasted significantly with that obtained after lipopolysaccharide (LPS) treatment, although splenic and hepatic tissues showed a similar distribution of TNF mRNA-positive cells. In the Colon 38 tumor, the action of LPS was limited to host cells in the periphery of the vessels. DMXAA treatment induced 7-fold higher peak TNF levels in tumor than in serum. In contrast, LPS treatment induced 9-fold higher TNF levels in serum than in tumor. DMXAA induced 35-fold higher TNF activity in the Colon 38 tissue than did LPS. One ovarian, one squamous, and three melanoma human tumor xenografts implanted in athymic nude mice expressed TNF mRNA of human and murine origin in response to DMXAA, confirming that DMXAA can activate both host and tumor cells. The use of low molecular weight agents to induce TNF synthesis in situ in the tumor represents a novel approach to TNF-mediated therapy of cancers.

Antitumour activity of the novel immune modulator 5,6-dimethylxanthenone-4-acetic acid (DMXAA) in mice lacking the interferon-gamma receptor.

5,6-Dimethylxanthenone-4-acetic acid (DMXAA), a novel antitumour agent currently undergoing clinical evaluation, appears to mediate its antitumour effects through immune modulation and the production of cytokines. We used mice with a targeted disruption of the interferon-gamma (IFN-gamma) receptor gene as a model to evaluate the role of the host response to IFN-gamma in the antitumour action of DMXAA on colon 38 tumours. A feature of the results was that while DMXAA treatment induced both IFN-gamma and tumour necrosis factor (TNF) in serum, the increase was > 20-fold higher in IFN-gamma R0/0 mice than in wild-type mice. In contrast, mRNA levels for IFN-gamma and TNF were similar in the two mouse strains, suggesting that the concentrations of these cytokines were controlled by a post-transcriptional mechanism. Serum nitrate levels, used as a measure of nitric oxide production, were increased by DMXAA, but to a similar extent in both strains of mice. Complete regressions of colon 38 tumours were obtained in response to DMXAA in the knockout mice, although the dose required for 100% cure was higher and the reduction in tumour volume occurred more slowly than in the wild-type counterparts. The results demonstrate that the host response to IFN-gamma is not essential for an anti-tumour response. Similar results were obtained in mice that were immunosuppressed by treatment with cyclosporin A before treatment with DMXAA. The results are consistent with the concept that the antitumour activity of DMXAA involves complex immunomodulation, probably with significant redundancy in contributing cytokines.

Production of tumour necrosis factor-alpha by cultured human peripheral blood leucocytes in response to the anti-tumour agent 5,6-dimethylxanthenone-4-acetic acid (NSC 640488).

The investigative anti-tumour agent 5,6-dimethylxanthenonone-4-acetic acid (DMXAA, NSC 640488), developed in this laboratory as an improved analogue of flavone acetic acid (FAA, NSC 347512), is currently in clinical trial. The ability of DMXAA to up-regulate tumour necrosis factor (TNF) mRNA and protein synthesis in cultured human peripheral blood leucocytes (HPBLs) has been investigated and compared with that of flavone acetic acid (FAA) and of bacterial lipopolysaccharide (LPS). Human peripheral blood leucocytes were isolated from buffy coats obtained from a blood transfusion centre and also from blood samples from laboratory volunteers. At a concentration of 400 microg ml(-1) and an incubation time of 2 h, DMXAA up-regulated mRNA synthesis in six of eight individuals tested, as measured by Northern blotting. The degree of up-regulation varied in different individuals from one to nine times that of control levels. In contrast, FAA caused no induction above that of control levels and in some cases suppressed expression relative to controls, extending previous data that DMXAA but not FAA up-regulates TNF mRNA in the human HL-60 tumour cell line. At the same concentration but with longer incubation times (6-12 h), DMXAA induced increases in TNF protein in 11 of 15 samples of HPBLs from buffy coats and also in 11 of 15 samples of HPBLs from volunteers, as measured by cytotoxicity assays with L929 cells. FAA caused no increase in TNF protein, while LPS induced TNF to approximately 20-fold higher levels than did DMXAA. Considerable heterogeneity of response was observed with both sources of HPBLs, and there was little or no correlation between the extent of TNF induction by DMXAA and LPS in individual samples. In vitro analysis of the response of human peripheral blood leucocytes to DMXAA may be a useful test in clinical trials of agents such as DMXAA.

Effect of thalidomide on tumour necrosis factor production and anti-tumour activity induced by 5,6-dimethylxanthenone-4-acetic acid.

The investigational anti-tumour agent, 5,6-dimethylxanthenone-4-acetic acid (5,6-MeXAA), an analogue of flavone acetic acid (FAA), has been scheduled for clinical evaluation. Like FAA, 5,6-MeXAA exhibits excellent experimental anti-tumour activity and is an efficient inducer of cytokines in mice. We have examined the effect of pharmacological suppression of tumour necrosis factor (TNF) production on the anti-tumour activity of 5,6-MeXAA, taking advantage of previous observations that TNF production in response to endotoxin in vitro is inhibited by thalidomide. Thalidomide at doses of between 8 and 250 mg kg-1 efficiently suppressed serum TNF activity in response to 5,6-MeXAA at its optimal TNF inducing dose of 55 mg kg-1. Suppression was achieved when thalidomide was administered at the same time as, or up to 4 h before, 5,6-MeXAA. Under conditions in which TNF activity was suppressed, the degree of tumour haemorrhagic necrosis and the proportion of cures in the subcutaneous Colon 38 tumour were increased. In mice administered thalidomide (100 mg kg-1) together with 5,6-MeXAA (30 mg kg-1), complete tumour regression was obtained in 100% of mice, as compared with 67% in mice receiving 5,6-MeXAA alone. The results suggest a possible new application for thalidomide and pose new questions about the action of 5,6-MeXAA and related compounds.

Induction of tumor necrosis factor-alpha messenger RNA in human and murine cells by the flavone acetic acid analogue 5,6-dimethylxanthenone-4-acetic acid (NSC 640488).

The investigational antitumor agent, 5,6-dimethylxanthenone-4-acetic acid (5,6-MeXAA; NSC 640488) induced greater expression of tumor necrosis factor-alpha (TNF-alpha) mRNA in murine spleen cells in vivo at its optimal dose of 27.5 mg/kg than flavone acetic acid (FAA; NSC 347512) at its optimal dose of 220 mg/kg. Up-regulation of TNF-alpha mRNA was obtained using 5,6-MeXAA in vitro in cultures of murine splenocytes, the murine J774 macrophage cell line, and the human HL-60 myelomonocytic leukemia cell line. Maximal induction occurred at a 5,6-MeXAA concentration of 200 micrograms/ml for both murine J774 and human HL-60 cells. A direct comparison of FAA and 5,6-MeXAA (100-600 micrograms/ml) to stimulate TNF-alpha mRNA in HL-60 cells showed activity by 5,6-MeXAA at all doses but minimal activity with FAA. The results demonstrate that 5,6-MeXAA is equally potent in up-regulating TNF-alpha mRNA in human and murine cells of the monocyte/macrophage lineage, whereas FAA has demonstrable activity in murine cells only. The results suggest that 5,6-MeXAA would be a more active clinical agent than FAA because TNF-alpha induction appears to be a critical factor in the antitumor effects of this class of compounds.