Combination of verteporfin-photodynamic therapy with 5-aza-2'-deoxycytidine enhances the anti-tumour immune response in triple negative breast cancer.

Introduction: Triple negative breast cancer (TNBC) is a subtype of breast cancer characterised by its high tumourigenic, invasive, and immunosuppressive nature. Photodynamic therapy (PDT) is a focal therapy that uses light to activate a photosensitizing agent and induce a cytotoxic effect. 5-aza-2'-deoxycytidine (5-ADC) is a clinically approved immunomodulatory chemotherapy agent. The mechanism of the combination therapy using PDT and 5-ADC in evoking an anti-tumour response is not fully understood. Methods: The present study examined whether a single dose of 5-ADC enhances the cytotoxic and anti-tumour immune effect of low dose PDT with verteporfin as the photosensitiser in a TNBC orthotopic syngeneic murine model, using the triple negative murine mammary tumour cell line 4T1. Histopathology analysis, digital pathology and immunohistochemistry of treated tumours and distant sites were assessed. Flow cytometry of splenic and breast tissue was used to identify T cell populations. Bioinformatics were used to identify tumour immune microenvironments related to TNBC patients. Results: Functional experiments showed that PDT was most effective when used in combination with 5-ADC to optimize its efficacy. 5-ADC/PDT combination therapy elicited a synergistic effect in vitro and was significantly more cytotoxic than monotherapies on 4T1 tumour cells. For tumour therapy, all types of treatments demonstrated histopathologically defined margins of necrosis, increased T cell expression in the spleen with absence of metastases or distant tissue destruction. Flow cytometry and digital pathology results showed significant increases in CD8 expressing cells with all treatments, whereas only the 5-ADC/PDT combination therapy showed increase in CD4 expression. Bioinformatics analysis of in silico publicly available TNBC data identified BCL3 and BCL2 as well as the following anti-tumour immune response biomarkers as significantly altered in TNBC compared to other breast cancer subtypes: GZMA, PRF1, CXCL1, CCL2, CCL4, and CCL5. Interestingly, molecular biomarker assays showed increase in anti-tumour response genes after treatment. The results showed concomitant increase in BCL3, with decrease in BCL2 expression in TNBC treatment. In addition, the treatments showed decrease in PRF1, CCL2, CCL4, and CCL5 genes with 5-ADC and 5-ADC/PDT treatment in both spleen and breast tissue, with the latter showing the most decrease. Discussion: To our knowledge, this is the first study that shows which of the innate and adaptive immune biomarkers are activated during PDT related treatment of the TNBC 4T1 mouse models. The results also indicate that some of the immune response biomarkers can be used to monitor the effectiveness of PDT treatment in TNBC murine model warranting further investigation in human subjects.

Ligand-bound integrin αvß6 internalisation and trafficking.

The integrin αvß6 is expressed at low levels in most normal healthy tissue but is very often upregulated in a disease context including cancer and fibrosis. Integrins use endocytosis and trafficking as a means of regulating their surface expression and thus their functions, however little is known of how this process is regulated in the context of αvß6. As αvß6 is a major target for the development of therapeutics in cancer and fibrosis, understanding these dynamics is critical in the development of αvß6-targeted therapies. Following development of a flow cytometry-based assay to measure ligand (A20FMDV2 or LAP)-bound αvß6 endocytosis, an siRNA screen was performed to identify which genes were responsible for internalising αvß6. These data identified 15 genes (DNM2, CBLB, DNM3, CBL, EEA1, CLTC, ARFGAP3, CAV1, CYTH2, CAV3, CAV2, IQSEC1, AP2M1, TSG101) which significantly decreased endocytosis, predominantly within dynamin-dependent pathways. Inhibition of these dynamin-dependent pathways significantly reduced αvß6-dependent migration (αvß6-specific migration was 547 ± 128 under control conditions, reduced to 225 ± 73 with clathrin inhibition, and 280 ± 51 with caveolin inhibition). Colocalization studies of αvß6 with endosome markers revealed that up to 6 h post-internalisation of ligand, αvß6 remains in Rab11-positive endosomes in a perinuclear location, with no evidence of αvß6 degradation up to 48 h post exposure to A20FMDV2. Additionally, 60% of ligand-bound αvß6 was recycled back to the surface by 6 h. With studies ongoing using conjugated A20FMDV2 to therapeutically target αvß6 in cancer and fibrosis, these data have important implications. Binding of A20FMDV2 seemingly removes much of the αvß6 from the cell membrane, and upon its recycling, a large fraction appears to still be in the ligand-bound state. While these results are observed with A20FMDV2, these data will be of value in the design of αvß6-specific therapeutics and potentially the types of therapeutic load.

Alternative tissue fixation for combined histopathological and molecular analysis in a clinically representative setting.

Formalin is the principal tissue fixative used worldwide for clinical and research purposes. Despite optimal preservation of morphology, its preservation of DNA and RNA is poor. As clinical diagnostics increasingly incorporates molecular-based analysis, the requirement for maintaining nucleic acid quality is of increasing importance. Here we assess an alternative non-formalin-based tissue fixation method, PAXgene Tissue system, with the aim of better preserving nucleic acids, while maintaining the quality of the tissue to be used for vital existing diagnostic techniques. In this study, these criteria are assessed in a clinically representative setting. In total, 203 paired PAXgene Tissue and formalin-fixed samples were obtained. Blind-scored haematoxylin and eosin (H&E) sections showed comparable and acceptable staining. Immunohistochemistry (IHC) staining was suboptimal using existing protocols but improved with minor method adjustment and optimisation. Quality of DNA and RNA was significantly improved by PAXgene tissue fixation [RIN 2.8 versus 3.8 (p < 0.01), DIN 5.68 versus 6.77 (p < 0.001)], which translated into improved performance on qPCR assay. These results demonstrate the potential of PAXgene Tissue to be used routinely in place of formalin, maintaining adequate histological staining and significantly improving the preservation of biological molecules in the genomic era.

Harnessing the power of foot-and-mouth-disease virus for targeting integrin alpha-v beta-6 for the therapy of cancer.

Introduction: The integrin αvß6 is a promising therapeutic target due to its limited expression in healthy tissue and significant overexpression in cancer and fibrosis. The peptide A20FMDV2, derived from the foot and mouth disease virus, is highly selective for αvß6, and can be used therapeutically to target αvß6 expressing cells.Areas covered: In this review, the authors discuss the logic that led to the discovery of A20FMDV2, the importance of its stereochemistry in receptor-binding, and the strategies employed to use it as a molecular-specific drug delivery system. These strategies include creating A20FMDV2-drug conjugates, genetically modifying oncolytic viruses to express A20FMDV2 and thus redirect their tropism to predominantly αvß6 expressing cells, creation of A20FMDV2 expressing CAR T-cells, and modifying antibody tropism by inserting A20FMDV2 into the CDR3 loop.Expert opinion: αvß6 is one of the most promising therapeutic targets in cancer and fibrosis discovered in the last few decades. The potential use of A20FMDV2 as a molecular-specific αvß6-targeting agent is extremely promising, particularly when considering the success of the peptide and its variants in clinical imaging.

The ITGB6 gene: its role in experimental and clinical biology.

Integrin αvß6 is a membrane-spanning heterodimeric glycoprotein involved in wound healing and the pathogenesis of diseases including fibrosis and cancer. Therefore, it is of great clinical interest for us to understand the molecular mechanisms of its biology. As the limiting binding partner in the heterodimer, the ß6 subunit controls αvß6 expression and availability. Here we describe our understanding of the ITGB6 gene encoding the ß6 subunit, including its structure, transcriptional and post-transcriptional regulation, the biological effects observed in ITGB6 deficient mice and clinical cases of ITGB6 mutations.

The ITGB6 gene: its role in experimental and clinical biology.

Integrin αvß6 is a membrane-spanning heterodimeric glycoprotein involved in wound healing and the pathogenesis of diseases including fibrosis and cancer. Therefore, it is of great clinical interest for us to understand the molecular mechanisms of its biology. As the limiting binding partner in the heterodimer, the ß6 subunit controls αvß6 expression and availability. Here we describe our understanding of the ITGB6 gene encoding the ß6 subunit, including its structure, transcriptional and post-transcriptional regulation, the biological effects observed in ITGB6 deficient mice and clinical cases of ITGB6 mutations.