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. Author manuscript; available in PMC: 2022 Aug 25.
Published in final edited form as: Cell Rep. 2022 Aug 16;40(7):111201. doi: 10.1016/j.celrep.2022.111201

Figure 2. The VCAN-matrikine versikine promotes cDC1 abundance in vivo.

Figure 2.

(A) Schematic of the experiment. LLC tumor cells were engineered to express hemagglutinin (HA)-tagged versikine (LLC-Vkine) or empty vector controls (LLC-EV) and injected subcutaneously (s.c.) on the flank or intravenously using a retro-orbital approach.

(B) Gross morphology of orthotopic (top) and s.c. (bottom) LLC-EV and LLC-Vkine tumors.

(C) Anti-HA tag western blotting detects a 75-kDa band in LLC-Vkine tumor lysates, consistent with versikine. See the full blot in Figure S2A.

(D) Representative immunohistochemistry (IHC) images showing α-DPEAAE and HA tag staining of LLC-EV and LLC-Vkine tumors. Endogenous DPEAAE proteolysis is low level and similar between LLC-EV and LLC-Vkine. Anti-HA staining localizes in a membranous distribution in LLC-Vkine cells (inset, larger magnification).

(E) Flow cytometric analysis of cDC subsets in s.c. LLC-EV and LLC-Vkine tumors (gating strategy according to Figure S1F) (Laoui et al., 2016) and quantification of cDC and tumor-associated DC (TADC) frequency (top) and absolute count ratios (cDC1/cDC1+cDC2 and cDC2/cDC1+cDC2) (bottom).

(F) Comparison of immune contexture (CD45+ fraction) in LLC-EV versus LLC-Vkine tumors by 31-marker mass cytometry.

(G) Flow cytometry analysis of cDC subsets in orthotopic LLC-EV and LLC-Vkine tumors (lung metastases induced by intravenous injection). A summary of cDC and TADC subset frequencies is depicted on the right.

Data are presented as mean ± SEM and are from one of three independent experiments with n = 5 or 6 for each group. *p < 0.05, **p < 0.01, ***p < 0.001. In vitro experiments were performed in technical triplicates. In vivo cohort sizes are shown in individual panels. All experiments were reproduced independently at least twice.