Journal: bioRxiv
Article Title: Ex vivo model of breast cancer cell invasion in live lymph node tissue
doi: 10.1101/2024.07.18.601753
Figure Lengend Snippet: Reduced invasion of cancer cells in pre-metastatic iTDLN ex vivo . (A) Schematic illustration of in vivo model of breast cancer from which TDLN were obtained. Bottom-up view of the animal. (B) Growth kinetics of BRPKp110 mammary tumors (n = 3 mice). (C) Flow cytometry analysis of cancer cell in TDLN. Quantification of CD45- anti-GFP+ cells in TDLNs 5 days post BRPKp110 inoculation. Mean ± stdev; each data point represents a fraction of CD45- anti-GFP+ cells per LN (n = 6 LNs/ group (inguinal, axillary) obtained from 3 tumor-bearing mice and 3 control mice injected with PBS). Two-way ANOVA, followed by Tukey posthoc test. p > 0.05. (D) Representative images of cancer cell invasion (WT BRPKp110, black) into control LN, pre-metastatic iTDLN and aTDLN at 1 hr and 20 hr post overlay. Scale bar 200 µm. (E) BRPKp110+ area positive area in control LNs, a non-tumor mice injected with PBS, pre- metastatic iTDLN and aTDLN after 1h and 20h of culture. Mean ± stdev; each data point represents an individual LN slice (n=2-3/group, LN slices obtained from 3 mice). Two-way ANOVA with Tukey posthoc test. *p < 0.05. (F) Concentrations of CCL21, CCL19, CXCL12 and CXCL13 in CM from TDLN slices after 20 hr culture, measured by ELISA. Mean ± stdev; each dot shows the supernatant from one LN slice (n = 8-10 slices, pooled from 3 female mice). Two-way ANOVA with Tukey posthoc test. *p < 0.05. (G) Migration of untreated and PT- treated BRPKp110 towards TDLN CM. Mean ± stdev; each data point represents the mean migration fold change per membrane, calculated from three non-overlapping fields of view (n = 3-4 membranes/condition; normalized data pooled from 3 independent in vitro experiments). Two-way ANOVA, followed by Tukey posthoc test. *p < 0.05, ***p < 0.001, ****p < 0.0001. (H) Intranodal levels of IL-21 in were significantly higher in pre-metastatic iTDLN and aTDLN than in control LN. Mean ± stdev; each data point represents the contents of 2 pooled LNs. Control: 12 LNs, 3 mice. iTDLN: 8 LNs, 4 mice, aTDLN: 10 LNs, 5 mice. Two-way ANOVA, followed by Tukey posthoc test. **p < 0.01, *p < 0.05.
Article Snippet: Samples were analyzed by sandwich ELISA assay using DuoSet ELISA development kit for Il-21 (catalog no. DY594; R&D Systems, Inc., Minneapolis, MN, USA).
Techniques: Ex Vivo, In Vivo, Flow Cytometry, Control, Injection, Enzyme-linked Immunosorbent Assay, Migration, Membrane, In Vitro
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