aav8 dio hm4di mcherry  (Addgene inc)

Journal: Nature

Article Title: Tumour–brain crosstalk restrains cancer immunity via a sensory–sympathetic axis

doi: 10.1038/s41586-025-10028-8

Figure Lengend Snippet: a , Experimental setup and representative immunofluorescence images showing anterogradely labelled Npy2r + or P2ry1 + vagal nerve fibres (red) around KP tumours (green). White arrows denote tumour-innervating nerve fibres. n = 3 mice per group. Scale bars: 500 μm (left), 100 μm (right). b , c , Experimental setup, representative haematoxylin and eosin (H&E)-stained lung sections and tumour quantification in Npy2r- IRES-cre; LSL-DTR ( b ) or P2ry1- IRES-cre; LSL-DTR ( c ) mice that received VNG injection of PBS or DT and were orthotopically transplanted with KP tumour cells. Lung tumour burden was assessed as the fraction of tumour area over total tissue area ( b : n = 16 per group, P < 0.0001; c : n = 8 PBS, n = 9 DT, P = 0.5919) and total lung mass ( b : n = 13 per group, P = 0.0001; c : n = 8 PBS, n = 9 DT, P = 0.2787). Scale bars, 1 mm. d , Experimental setup and representative immunofluorescence images showing anterogradely labelled Trpv1 + vagal nerve fibres (red) around KP tumours (green). White arrows denote tumour-innervating nerve fibres. n = 3 mice per group. Scale bar: 500 μm (left), 100 μm (right). e , Experimental setup, representative H&E-stained lung sections and tumour quantification in Trpv1-cre; LSL-DTR mice that received VNG injection of PBS or DT and were orthotopically transplanted with KP tumour cells. Lung tumour burden was assessed by tumour/tissue area ( n = 10 PBS, n = 12 DT, P < 0.0001) and total lung mass ( n = 20 per group, P < 0.0001). Scale bars, 1 mm. In b , c , e , Tumour/tissue area and total lung mass for individual mice are expressed relative to mean of the corresponding PBS-treated group within each cohort (pooled from 2 to 3 independent experiments). f , Experimental setup, representative H&E-stained lung sections, and tumour quantification in KP; Trpv1 DTR+ ( n = 7) or KP; Trpv1 DTR − ( n = 11) mice injected intratracheally (IT) with adSPC-cre to initiate tumours, injected with DT in the VNG 3 weeks after tumour induction, and tissue collected 13 weeks post tumour initiation. Tumour/tissue area P = 0.0248, total lung mass P = 0.0236, tumour grade P = 0.0060. Scale bars, 1 mm. g , Experimental scheme, representative H&E section and tumour quantification from Trpv1-cre mice injected intratracheally with retro-AAV-hSyn-flex-hM4Di-mCherry (hM4Di, n = 7) or retro-AAV-hSyn-flex-mCherry (control, n = 8), and treated with CNO (intraperitoneal (IP) injection). Tumour/tissue area P = 0.0150, total lung mass P = 0.0151. Scale bars, 1 mm. In f , g , results shown are from one experiment, representative of two independent experiment. Data are expressed as mean ± s.e.m. Unpaired, two-tailed Student’s t -test ( b , c , e – g ).

Article Snippet: For chemogenetic silencing of lung-innervating VSNs, 5 μl of AAVretro-hSyn-flex-mCherry (Addgene 50459-AAVrg), or AAVretro-hSyn-flex-hM4Di-mCherry (Addgene 44362-AAVrg) was mixed with 45 μl of PBS and intratracheally injected to mice.

Techniques: Immunofluorescence, Staining, Injection, Control, Two Tailed Test

Journal: Nature

Article Title: Tumour–brain crosstalk restrains cancer immunity via a sensory–sympathetic axis

doi: 10.1038/s41586-025-10028-8

Figure Lengend Snippet: a , b , Representative images and quantification of Slc17a6 (red) and Fos (cyan) by RNAscope in RVLM neurons from healthy ( n = 5), KP tumour-bearing Trpv1-cre; LSL-DTR ( a ) or Npy2r- IRES-cre; LSL-DTR ( b ) mice that were injected with PBS or DT in the VNG ( a : n = 7 PBS, n = 5 DT, P < 0.0001 for PBS versus healthy and PBS versus DT; b : n = 5 PBS, n = 6 DT, P < 0.0001). Scale bars, 100 μm. c , Representative immunofluorescence images and quantification of FOS + neurons in T3–5 thoracic ganglia from healthy ( n = 4) or tumour-bearing Npy2r- IRES-cre; LSL-DTR mice injected with PBS ( n = 4) or DT ( n = 4) in the VNG. P < 0.0001 for PBS versus healthy and PBS versus DT. Scale bars, 50 μm. d , Experimental setup (top), representative H&E-stained lung sections (bottom left) and quantification of lung tumour burden (bottom right) from Vglut2-cre mice injected with AAV-DIO-mCherry (control, n = 6) or AAV-DIO-hM4Di (hM4Di, n = 5) in the RVLM. P = 0.0062. Scale bars, 1 mm. e , Representative immunofluorescence images and quantification of TH + sympathetic nerves (green) around KP tumours (red) from Trpv1-cre; LSL-DTR mice that received VNG injection of PBS or DT. The number of TH + nerves around each tumour ( n = 153 PBS, n = 75 DT, P < 0.0001) as well as the average number of TH + nerve fibres per tumour in each mouse ( n = 13 PBS, n = 10 DT, P = 0.0011) were quantified. 5.1× and 1.3× refer to the fold change between indicated groups. Scale bars, 100 μm. f , Noradrenaline levels in tumour-bearing lung tissue from Npy2r- IRES-cre; LSL-DTR mice that received VNG injection of PBS ( n = 5) or DT ( n = 6). P = 0.0165 by tissue mass, P = 0.0044 by lung lobe. g , Quantification of VAChT + parasympathetic nerves around tdTomato + tumours from Trpv1-cre; LSL-DTR mice that received VNG injection of PBS or DT. The number of VAChT + nerves around each tumour ( n = 95 PBS, n = 40 DT) as well as the average number of VAChT + nerve fibres per tumour in each mouse ( n = 7 PBS, n = 5 DT) were quantified. Results shown are representative of two independent experiments. Data are expressed as mean ± s.e.m. One-way ANOVA with Tukey’s multiple comparisons ( a , c ) or unpaired, two-tailed Student’s t -test ( b , d – g ) was performed. Brain cartoons in a , b , d were created in BioRender. Jin, C. (2026) https://BioRender.com/9zdoahw .

Article Snippet: For chemogenetic silencing of lung-innervating VSNs, 5 μl of AAVretro-hSyn-flex-mCherry (Addgene 50459-AAVrg), or AAVretro-hSyn-flex-hM4Di-mCherry (Addgene 44362-AAVrg) was mixed with 45 μl of PBS and intratracheally injected to mice.

Techniques: RNAscope, Injection, Immunofluorescence, Staining, Control, Two Tailed Test