3D Microfluidic model for evaluating immunotherapy efficacy by tracking dendritic cell behaviour toward tumor cells

Immunotherapy efficacy relies on the crosstalk within the tumor microenvironment between cancer and dendritic cells (DCs) resulting in the induction of a potent and effective antitumor response. DCs have the specific role of recognizing cancer cells, taking up tumor antigens (Ags) and then migrating to lymph nodes for Ag (cross)-presentation to naïve T cells. Interferon-α-conditioned DCs (IFN-DCs) exhibit marked phagocytic activity and the special ability of inducing Ag-specific T-cell response. Here, we have developed a novel microfluidic platform recreating tightly interconnected cancer and immune systems with specific 3D environmental properties, for tracking human DC behaviour toward tumor cells. By combining our microfluidic platform with advanced microscopy and a revised cell tracking analysis algorithm, it was possible to evaluate the guided efficient motion of IFN-DCs toward drug-treated cancer cells and the succeeding phagocytosis events. Overall, this platform allowed the dissection of IFN-DC-cancer cell interactions within 3D tumor spaces, with the discovery of major underlying factors such as CXCR4 involvement and underscored its potential as an innovative tool to assess the efficacy of immunotherapeutic approaches

Impaired IFN-α-mediated signal in dendritic cells differentiates active from latent tuberculosis

<div><p>Individuals exposed to <i>Mycobacterium tuberculosis</i> (<i>Mtb</i>) may be infected and remain for the entire life in this condition defined as latent tuberculosis infection (LTBI) or develop active tuberculosis (TB). Among the multiple factors governing the outcome of the infection, dendritic cells (DCs) play a major role in dictating antibacterial immunity. However, current knowledge on the role of the diverse components of human DCs in shaping specific T-cell response during <i>Mtb</i> infection is limited. In this study, we performed a comparative evaluation of peripheral blood circulating DC subsets as well as of monocyte-derived Interferon-α DCs (IFN-DCs) from patients with active TB, subjects with LTBI and healthy donors (HD). The proportion of circulating myeloid BDCA3⁺ DCs (mDC2) and plasmacytoid CD123⁺ DCs (pDCs) declined significantly in active TB patients compared to HD, whereas the same subsets displayed a remarkable activation in LTBI subjects. Simultaneously, the differentiation of IFN-DCs from active TB patients resulted profoundly impaired compared to those from LTBI and HD individuals. Importantly, the altered developmental trait of IFN-DCs from active TB patients was associated with down-modulation of IFN-linked genes, marked changes in molecular signaling conveying antigen (Ag) presentation and full inability to induce Ag-specific T cell response. Thus, these data reveal an important role of IFN-α in determining the induction of <i>Mtb-</i>specific immunity.</p></div

IFN-DCs derived from active TB patients are impaired for DC-related response gene signatures.

<p><i>A</i>, GSEA results for comparison between active TB and HD. GeneSets relevant to DC biology are shown. Plot of the running sum for enrichment score (ES) in selected data set, the normalized enrichment score (NES) and location of genes (hits) in the list ranked according to expression differences (barcode) are shown as explained in the legend. <i>B</i>, Dot plot representation of GeneSet expression level in comparisons among active TB, LTBI and HD groups, as expressed by NES and q-value. As detailed in the legend, dot area is proportional to NES (<1 = no enrichment; >2 = maximum expression); dot color intensity is proportional to q-value significance, dot color indicates the group in which the GeneSet is overexpressed.</p

Analysis of differentially expressed genes across active TB, LTBI and HD individuals.

<p>Hereafter, active TB, LTBI and HD indicate TB-DCs, LTBI-DCs and HD-DCs, respectively. <i>A</i>, Venn’s diagram showing the number of transcripts modulated and their overlaps across IFN-DCs derived from active TB, LTBI and HD individuals. Genes selected on the threshold of <i>P</i> <0.005 by Student’s t-test from five biological replicates (n = 5) for each group were included in the analysis. <i>B</i>, PCA of differentially expressed genes across active TB, LTBI and HD; PC1 and PC2 were chosen as the axes explicating most of the data variance. <i>C</i>, Heat map showing differential expression across active TB, LTBI and HD, built on the selected 170 genes differentially expressed between active TB and HD individuals. The expression level average of five samples for each group is shown. Values are average corrected and shown on a blue-black-yellow gradient: yellow, increased expression; blue, decreased expression.</p

IPA analysis identifies specific impaired functional terms in IFN-DCs derived from active TB patients.

<p>Gene lists of 1.5-fold differentially expressed genes in TB-DCs <i>versus</i> HD-DCs were mapped by IPA software, revealing the modulation of several pathways and genes. <i>A</i>, Genes forming “activation dendritic cells” and “apoptosis dendritic cells” annotation found to be affected in TB-DCs. The direction of change is color coded with green and red indicating down-regulation and up-regulation, respectively. <i>B</i>, Regulator Effect Analysis of TB-DCs indicating upstream regulators (at the top) and downstream effects (at the bottom) hypothesized by IPA based on expression level of key genes (in the middle). Color legend as in <i>A</i>. <i>C</i>, Network of genes most significantly altered in TB-DCs. Genes in green showed a 1.5 fold down-regulation compared to HD-DCs.</p

Analysis of circulating DC subsets in peripheral blood of active TB, LTBI or HD individuals.

<p><i>A</i>, Percentage of peripheral blood Lin^-/HLA-DR⁺ DCs, CD11c⁺ mDC1, BDCA3⁺ mDC2 and CD123⁺ pDCs were determined by flow cytometry. Median values are shown. <i>B</i>, CD86 and CD80 expression on total DCs and DC subsets are shown. Data are reported as mean fluorescence intensity (MFI) and median values are indicated. Statistical significance was analyzed by Mann-Whitney test using Bonferroni correction. *<i>P</i><0.05; **<i>P</i><0.01; ***<i>P</i><0.001; ****<i>P</i><0.0001. <i>C</i>, Expression of the type-1 and type-2 myeloid DC markers, BDCA1 and BDCA3, and the plasmacytoid marker CD123 in IFN-DCs derived from active TB, LTBI and HD individuals by flow cytometry. Active TB, LTBI and HD indicate IFN-DCs derived from active TB, LTBI and HD individuals, respectively. Percentages of positive cells are shown. SSC, side scatter. Data are representative of three independent experiments (N = 3).</p