Onychomycosis is predominantly caused by the dermatophytes Trichophyton rubrum, Trichophyton mentagrophytes and Trichophyton tonsurans. The main treatment obstacle concerns low nail-plate drug permeability. In vitro antifungal photodynamic treatment (PDT) and nail penetration enhancing effectiveness have been proven for multifunctional photosensitizer 5,10,15-tris(4-N-methylpyridinium)-20-(4-(butyramido-methylcysteinyl)-hydroxyphenyl)-[21H,23H]-porphine trichloride (PORTHE). This study investigates single PORTHE green laser/LED PDT of varying degrees of ex vivo onychomycoses in a human nail model. T. mentagrophytes, T. rubrum, T. tonsurans onychomycoses were ex vivo induced on nail pieces at 28 °C (normal air) and 37 °C (6.4% CO2) during 3 to 35 days and PDTs applied to the 37 °C infections. All dermatophytes showed increasingly nail plate invasion at 37 °C between 7 and 35 days; arthroconidia were observed after 35 days for T. mentagrophytes and T. tonsurans. Using 81 J/cm2 (532 nm) 7-day T. mentagrophytes onychomycoses were cured (92%) with 80 µM PORTHE (pH 8) after 24 h propylene glycol (PG, 40%) pre-treatment and 35-day onychomycoses (52%–67%) with 24 h PORTHE (40–80 µM)/40% PG treatment (pH 5). 28 J/cm2 LED light (525 ± 37 nm) improved cure rates to 72%, 83% and 73% for, respectively, T. mentagrophytus, T. rubrum and T. tonsurans 35-day onychomycoses and to 100% after double PDT. Data indicate PDT relevance for onychomycosis.

Multiple myeloma is a disease of malignant plasma cells residing in the bone marrow, where interactions with local immune cells are thought to contribute to disease pathobiology. However, since a multiple myeloma diagnosis is virtually always preceded by an asymptomatic precursor phase, identifying early alterations in the bone marrow micro-environment following occupation by multiple myeloma cells remains challenging. Here we used the 5TGM1 transfer model of murine myeloma in combination with myeloma-permissive KaLwRij mice and myeloma-resistant C57Bl/6 mice and hypothesized that differential sensitivity to myeloma in these HLA-identical mouse strains has an immunological basis and might allow for dissection of early immune responses to myeloma cells. Using flow cytometry and single-cell RNA sequencing we show that C57Bl/6 mice can restrain tumor growth for prolonged periods, associated with activation of cytotoxic immune responses that were absent from KaLwRij mice. Transcriptional analysis of immune cells and stromal cells identified a central role for IFN-signaling in tumor containment, and antibody-mediated neutralization of IFNγ increased both incidence and outgrowth of multiple myeloma in C57Bl/6 mice. Together these findings highlight the ability of a fully functional immune system to control multiple myeloma progression in an IFNγ-dependent manner and suggest that transfer of 5TGM1 cells into parental C57Bl/6 mice can serve as a faithful model to track anti-myeloma immune responses in immune competent and genetically modifiable mice.

Introduction The introduction of new treatment regimens has significantly increased the progression free survival (PFS) of newly diagnosed multiple myeloma (MM) patients. However, even with these novel treatments, for some the disease remains refractory, highlighting the need to identify the pathobiology of high-risk MM. In MM patients, high levels of circulating tumor cells (CTCs) is associated with an inferior prognosis independent of high-risk cytogenetics (Chakraborty et al., 2016), suggesting that CTC numbers are a relevant reflection of tumor cell biology. We hypothesized that high levels of CTCs in MM patients are either the result of a transcriptionally distinct tumor clone with enhanced migration capacities, or driven by transcriptional differences present in the bone marrow (BM) tumor cells. To test these hypotheses, we 1) compared MM cells from paired blood and BM samples, and 2) compared BM tumor cells of patients with high and low CTC levels, using single cell RNA-sequencing. Results We isolated plasma cell (PCs) from viably frozen mononuclear cells of paired peripheral blood (PB) and BM aspirates from five newly diagnosed MM patients (0.5%-8% CTCs) to determine the presence of a distinct CTC subclone. We generated single cell transcriptomes from 44,779 CTCs and 35,697 BM PCs. In the total 9 clusters common to BM PCs and CTCs were identified upon single cell data integration, but no cluster specific for either source was detected. Only 25 genes were significantly differential expressed between CTCs and BM PCs. The absence of transcriptional clusters unique to either CTCs or BM PCs, and the transcriptional similarity between these two anatomical sites makes it highly unlikely that CTC levels are driven by the presence of a transcriptionally-primed migratory clone. We next set out to identify possible transcriptional differences in BM PCs from eight patients with high (2-22%) versus thirteen patients with low (0.004%-0.08%) percentages of CTCs. Recurrent high-risk mutations were present in both groups. Single cell transcriptomes were generated from 74,830 BM PCs. Single cell data integration across all patients led to the identification of 8 distinct PC clusters, one of which was characterized by enhanced proliferation as defined by STMN1 and MKI67 transcription. Interestingly, this proliferative cluster was increased in patients with a high percentage of CTCs. Furthermore, cell cycle analyses based on canonical G2M and S phase markers revealed that actively cycling PCs were more frequent in the BM of patients with a high percentage of CTCs (64% versus 30%, p<0.001), irrespective of the transcriptional cluster of origin. We hypothesized that plasma cell-extrinsic cues from the bone marrow micro-environment might be driving tumor proliferation. In order to substantiate this, we isolated BM immune cells from the same 21 patients and generated a library of 301,045 single immune cell transcriptomes. This library contained all major immune cell subsets, including CD4 + and CD8 + T cells, NK cells, B cells and monocytes. Comparative analyses of these cell populations in patients with either high or low levels of CTC are ongoing. Conclusion Through single cell transcriptomic analyses, we demonstrate that CTCs and BM PCs are transcriptionally similar. Importantly, we identify increased BM PC proliferation as a significant difference between patients with high and low levels of CTCs, implicating an increased tumor proliferation as one of the potential mechanisms driving CTC levels and MM disease pathobiology. The relation of the BM immune micro-environment to this altered proliferative state is currently under investigation. Disclosures van der Velden: Janssen: Other: Service Level Agreement; BD Biosciences: Other: Service Level Agreement; Navigate: Other: Service Level Agreement; Agilent: Research Funding; EuroFlow: Other: Service Level Agreement, Patents & Royalties: for network, not personally. Sonneveld: SkylineDx: Honoraria, Research Funding; Karyopharm: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding; Celgene/BMS: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding. Broyl: Sanofi: Honoraria; Janssen Pharmaceuticals: Honoraria; Celgene: Honoraria; Bristol-Meyer Squibb: Honoraria; Amgen: Honoraria.

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