ABSTRACT
In this study the effect of repeated-fractional intradermal administration of diphtheria toxoid (DT) compared to a single administration in the presence or absence of adjuvants formulated in dissolving microneedles (dMNs) was investigated. Based on an adjuvant screening with a hollow microneedle (hMN) system, poly(I:C) and gibbsite, a nanoparticulate aluminum salt, were selected for further studies: they were co-encapsulated with DT in dMNs with either a full or fractional DT-adjuvant dose. Sharp dMNs were prepared regardless the composition and were capable to penetrate the skin, dissolve within 20 min and deposit the intended antigen-adjuvant dose, which remained in the skin for at least 5 h. Dermal immunization with hMN in repeated-fractional dosing (RFrD) resulted in a higher immune response than a single-full dose (SFD) administration. Vaccination by dMNs led overall to higher responses than hMN but did not show an enhanced response after RFrD compared to a SFD administration. Co-encapsulation of the adjuvant in dMNs did not increase the immune response further. Immunization by dMNs without adjuvant gave a comparable response to subcutaneously injected DT-AlPO4 in a 15 times higher dose of DT, as well as subcutaneous injected DT-poly(I:C) in a similar DT dose. Summarizing, adjuvant-free dMNs showed to be a promising delivery tool for vaccination performed in SFD administration.
Subject(s)
Diphtheria Toxoid/administration & dosage , Drug Delivery Systems/methods , Microinjections/methods , Needles , Off-Label Use , Vaccination/methods , Adjuvants, Immunologic/administration & dosage , Adjuvants, Immunologic/metabolism , Animals , Diphtheria Toxoid/metabolism , Dose-Response Relationship, Drug , Drug Delivery Systems/instrumentation , Drug Evaluation, Preclinical/methods , Female , Humans , Injections, Intradermal/instrumentation , Injections, Intradermal/methods , Mice , Mice, Inbred BALB C , Microinjections/instrumentation , Skin/drug effects , Skin/metabolism , Vaccination/instrumentationABSTRACT
Enhanced epidermal growth factor receptor (EGFR) activity has been strongly linked to breast cancer progression and mediators of EGFR endocytosis may well be involved. We developed a semi-automated high-content fluorescence microscopy-based EGFR endocytosis screen to identify proteins that mediate EGFR endocytosis in human HBL100 breast cancer cells. Knockdown of 172 individual endocytosis and actin-regulatory genes with small interfering RNAs led to the identification of 14 genes of which the contribution to EGFR endocytosis in breast cancer is until now poorly defined, including DNAJC6, GDI2, FGD6, HAX1, NECAP2 and AnxA2. We show that depletion of the actin and endocytosis regulatory protein annexin A2 (AnxA2) in a panel of four triple negative breast cancer (TNBC) cell lines affected EGFR endocytosis. Depletion of AnxA2 in the aggressive and highly metastatic MDA-MB-231 TNBC cell line resulted in the inhibition of EGFR transport beyond the early endosomes. This inhibition coincided with enhanced epidermal growth factor (EGF)-induced cell migration and downstream signaling via c-Jun N-terminal kinase (JNK) and Akt. Moreover, AnxA2 knockdown increased lung metastasis formation in mice. The effect of AnxA2 knockdown on EGFR endocytosis in MDA-MB-231 was related to dephosphorylation/activation of the actin-severing protein cofilin, as re-expression of an inactive S3E-cofilin mutant, but not an active S3A-cofilin mutant, re-established EGFR endocytosis to control levels. Together, our data provide evidence for AnxA2 as a mediator of EGFR endocytosis and signaling in breast cancer via regulation of cofilin activation.
Subject(s)
Actin Depolymerizing Factors/metabolism , Annexin A2/metabolism , Endocytosis , ErbB Receptors/metabolism , Neoplasm Metastasis , Signal Transduction , Animals , Annexin A2/genetics , Cell Line, Tumor , Gene Knockdown Techniques , Humans , Mice , Microscopy, Fluorescence , RNA InterferenceABSTRACT
We have recently shown that the adaptor protein p140Cap regulates tumor properties in terms of cell motility and growth. Here, by using the highly metastatic rat adenocarcinoma cell line MTLn3-epidermal growth factor receptor (EGFR), we assess the role of p140Cap in metastasis formation. Orthotopic transplantation of MTLn3-EGFR cells over-expressing p140Cap in Rag2(-/-)γ(c)(-/-) mice resulted in normal primary tumor growth compared with the controls. Strikingly, p140Cap over-expression causes an 80% inhibition in the number of lung metastases. p140Cap over-expressing cells display a 50% reduction in directional cell migration, an increased number and size of focal adhesions, and a strong impairment in the ability to invade in a 3D matrix. p140Cap over-expression affects EGFR signaling and tyrosine phosphorylation of cortactin in response to EGF stimulation. Intriguingly, p140Cap associates with cortactin via interaction with its second proline-rich domain to the cortactin SH3 domain. The phosphomimetic cortactin tyrosine 421 mutant rescues migration and invasive properties in p140Cap over-expressing cells. Taken together, these data demonstrate that p140Cap suppresses the invasive properties of highly metastatic breast carcinoma cells by inhibiting cortactin-dependent cell motility.
