Journal: Developmental cell

Article Title: Bidirectional activation of stem-like programs between metastatic cancer and alveolar type 2 cells within the niche.

doi: 10.1016/j.devcel.2024.05.020

Figure Lengend Snippet: Figure 2. Metastatic competent cells activate distinct tissue stemness programs (A) Hematoxylin and eosin (H&E) stains of lung sections 2 weeks after intravenous injection of the indicated tumor cells (4T1, CT26, and 4T07). Table shows the efficiency of each model in generating lung metastasis in syngeneic BALB/c mice. (B) Quantification of the number of lung metastases in each mouse. Data are presented as mean ± SEM of two independent experiments containing 4–5 mice each. (C) Table indicating the average number of metastases per mouse in three different histological levels. (D) Quantification showing the average area of metastasis per mouse. Data are presented as mean ± SEM, as in Figure 2B. (E) Experimental design for isolation of lung epithelial cells (CD45CD31EpCAM+) from the distal lung (mCherry) or the metastatic niche (mCherry+) using GFP+

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Deoxyribonuclease I from bovine pancreas Sigma-Aldrich Cat# DN25-100MG 4’,6-diamidino-2-phenylindole (DAPI) Sigma-Aldrich Cat# D9542 Fetal bovine serum Labtech Cat# FB-1001/100-500 Penicillin-Streptomycin Thermo Fisher Scientific Cat# 15070063 Trypsin-EDTA (0.25%), phenol red Thermo Fisher Scientific Cat# 25200056 HBSS, calcium/magnesium-free, no phenol red Thermo Fisher Scientific Cat# 14175095 DMEM Thermo Fisher Scientific Cat# 41965062 DMEM/F12 Thermo Fisher Scientific Cat# 11330032 Advanced DMEM/F12 Thermo Fisher Scientific Cat# 12634-010 Human EGF Thermo Fisher Scientific Cat# PHG0313 B-27 Supplement Thermo Fisher Scientific Cat# 17504044 Insulin solution human Sigma-Aldrich Cat# I9278 Insulin-Transferrin-Selenium (ITS) Thermo Fisher Scientific Cat# 41400045 GlutaMAX Supplement Thermo Fisher Scientific Cat# 35050061 Polybrene Sigma-Aldrich Cat# TR-1003-G Puromycin dihydrochloride APExBIO Cat# B7587 Antigenfix Diapath Cat# P0014 Zombie Aqua BioLegend Cat# 423101 ECL Chemiluminescence Detection Reagent Cytiva Cat# 10308449 D-luciferin PerkinElmer Cat# 122799 Critical commercial assays PowerUp SYBR Green Master Mix Thermo Fisher Scientific Cat# A25742 SuperScript IV VILO with ezDNase Thermo Fisher Scientific Cat# 11766050 LS columns Miltenyi Biotec Cat# 130-042-401 RNeasy Mini Kit QIAGEN Cat# 74904 RNeasy Micro Kit QIAGEN Cat# 74004 Deposited data RNA-seq This study GSE240040 ATAC-seq This study GSE240040 Experimental models: Cell lines 4T1 ATCC Cat# CRL-2539 CT26 ATCC Cat# CRL-2638 4T07 The Francis Crick Institute N/A TS1 Gift from J. Joyce17 N/A B16F10 ATCC Cat# CRL-6475 MLE12 ATCC Cat# CRL-2110 MLg ATCC Cat# CCL-206 Experimental models: Organisms/strains Mouse: FVB/n The Francis Crick Institute Jax Strain # 001800 Mouse: BALB/c The Francis Crick Institute Jax Strain # 000651 Mouse: Sftpc-CreERT2;R26REYFP C57BL/6 Gift from J-H. Lee N/A Mouse: Actin-GFP FVB/n Gift from J. Huelsken N/A Mouse: tdTomato C57BL/6 The Francis Crick Institute N/A Mouse: MMTV-PyMT FVB/n The Francis Crick Institute Jax Strain # 022974 Mouse: MMTV-PyMT C57BL/6 The Francis Crick Institute N/A Mouse: Actin-GFP MMTV-PyMT FVB/n The Francis Crick Institute N/A Mouse: TSLP KO FVB/n The Francis Crick Institute N/A (Continued on next page) e2 Developmental Cell 59, 1–16.e1–e8, September 23, 2024

Techniques: Injection, Isolation

Journal: Developmental cell

Article Title: Bidirectional activation of stem-like programs between metastatic cancer and alveolar type 2 cells within the niche.

doi: 10.1016/j.devcel.2024.05.020

Figure Lengend Snippet: Figure 3. Chromatin accessibility of niche AT2 cells reflects their reprogrammed state (A) Flow cytometry plots indicating AT2 cells in the distal lung or the metastatic niche. (B) Principal component analysis (PCA) of ATAC-seq data of distal and niche AT2 cells, as indicated by their respective colors. (C) Chromatin accessibility Z score for the differentially expressed peaks (FDR % 0.05) of all four conditions. (D) Alluvial diagram of differentially accessible regions and how they are redistributed between distal and niche AT2 cells from 4T1 metastases. (E) Alluvial diagram of differentially accessible regions and how they are redistributed between niche AT2 cells from CT26 and 4T1 metastases. Motif enrichment analysis performed between CT26 and 4T1 niche AT2 cells highlighting a few of the transcription factors identified. (F) Representative immunofluorescence image of metastatic MMTV-PyMT tumor stained with DAPI (blue), the AT2 lineage marker YFP (green), KRT8 (white), and TFAP4 (red). Scale bars, 50 mm for entire metastatic loci and 10 mm for selected niche and distal areas. (G) Quantification of the mean fluorescence intensity of nuclear TFAP4 in distal and niche AT2 cells of MMTV-PyMT tumors. Data are from one of two independent experiments shown as mean ± SEM.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Deoxyribonuclease I from bovine pancreas Sigma-Aldrich Cat# DN25-100MG 4’,6-diamidino-2-phenylindole (DAPI) Sigma-Aldrich Cat# D9542 Fetal bovine serum Labtech Cat# FB-1001/100-500 Penicillin-Streptomycin Thermo Fisher Scientific Cat# 15070063 Trypsin-EDTA (0.25%), phenol red Thermo Fisher Scientific Cat# 25200056 HBSS, calcium/magnesium-free, no phenol red Thermo Fisher Scientific Cat# 14175095 DMEM Thermo Fisher Scientific Cat# 41965062 DMEM/F12 Thermo Fisher Scientific Cat# 11330032 Advanced DMEM/F12 Thermo Fisher Scientific Cat# 12634-010 Human EGF Thermo Fisher Scientific Cat# PHG0313 B-27 Supplement Thermo Fisher Scientific Cat# 17504044 Insulin solution human Sigma-Aldrich Cat# I9278 Insulin-Transferrin-Selenium (ITS) Thermo Fisher Scientific Cat# 41400045 GlutaMAX Supplement Thermo Fisher Scientific Cat# 35050061 Polybrene Sigma-Aldrich Cat# TR-1003-G Puromycin dihydrochloride APExBIO Cat# B7587 Antigenfix Diapath Cat# P0014 Zombie Aqua BioLegend Cat# 423101 ECL Chemiluminescence Detection Reagent Cytiva Cat# 10308449 D-luciferin PerkinElmer Cat# 122799 Critical commercial assays PowerUp SYBR Green Master Mix Thermo Fisher Scientific Cat# A25742 SuperScript IV VILO with ezDNase Thermo Fisher Scientific Cat# 11766050 LS columns Miltenyi Biotec Cat# 130-042-401 RNeasy Mini Kit QIAGEN Cat# 74904 RNeasy Micro Kit QIAGEN Cat# 74004 Deposited data RNA-seq This study GSE240040 ATAC-seq This study GSE240040 Experimental models: Cell lines 4T1 ATCC Cat# CRL-2539 CT26 ATCC Cat# CRL-2638 4T07 The Francis Crick Institute N/A TS1 Gift from J. Joyce17 N/A B16F10 ATCC Cat# CRL-6475 MLE12 ATCC Cat# CRL-2110 MLg ATCC Cat# CCL-206 Experimental models: Organisms/strains Mouse: FVB/n The Francis Crick Institute Jax Strain # 001800 Mouse: BALB/c The Francis Crick Institute Jax Strain # 000651 Mouse: Sftpc-CreERT2;R26REYFP C57BL/6 Gift from J-H. Lee N/A Mouse: Actin-GFP FVB/n Gift from J. Huelsken N/A Mouse: tdTomato C57BL/6 The Francis Crick Institute N/A Mouse: MMTV-PyMT FVB/n The Francis Crick Institute Jax Strain # 022974 Mouse: MMTV-PyMT C57BL/6 The Francis Crick Institute N/A Mouse: Actin-GFP MMTV-PyMT FVB/n The Francis Crick Institute N/A Mouse: TSLP KO FVB/n The Francis Crick Institute N/A (Continued on next page) e2 Developmental Cell 59, 1–16.e1–e8, September 23, 2024

Techniques: Flow Cytometry, Staining, Marker

Journal: Developmental cell

Article Title: Bidirectional activation of stem-like programs between metastatic cancer and alveolar type 2 cells within the niche.

doi: 10.1016/j.devcel.2024.05.020

Figure Lengend Snippet: Figure 4. Metastatic niche components influence AT2 cell behavior ex vivo (A)FlowcytometryplotsofdistalandnicheCD64+macrophages.CellsgatedonCD45+Ly6GCD64+werefurtheranalyzedforisolationofalveolar(Siglec-F+CD11low) or interstitial macrophages (Siglec-FCD11+). (B) Quantification of CD64+ macrophages in the metastatic niche (4T1) compared with the distal lung (n = 11 mice). (C) Representative bright-field images of FACS-sorted macrophages. Scale bars, 200 mm. (D) Schematic illustrating the generation of lung organoids with naive AT2 cells, MLg lung fibroblasts, and macrophages (mCherry+ or mCherry). (E) Organoid-forming efficiency of AT2 cells growing with or without macrophages from the distal lung or the metastatic niche of 4T1 or CT26 tumors. Data are presented as mean ± SEM and were generated from two independent experiments. (F) Quantification of the type/morphology of lung organoids. Data are presented as in Figure 4E, with 107 control organoids, 162 4T1 distal organoids, 133 4T1 niche organoids, 139 CT26 distal organoids, and 146 CT26 niche organoids.. (G) Quantification of the percentage of hollow alveolar organoids and data are presented as in Figure 4E. (H) Representative bright-field images of lung organoids in co-culture with macrophages. Scale bars, 500 mm. (I) Immunofluorescence image of lung organoids stained with DAPI (blue), Sftpc (green), and Sox2 (red). Alveolar, hollow alveolar, and bronchioalveolar organoids are indicated. Scale bars, 100 mm. (J) Fluorescence images of FACS-sorted distal and niche fibroblasts (CD45CD31EpCAMGFP+) from TS1 tumors growing in actin-GFP mice. Scale bars, 200 mm.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Deoxyribonuclease I from bovine pancreas Sigma-Aldrich Cat# DN25-100MG 4’,6-diamidino-2-phenylindole (DAPI) Sigma-Aldrich Cat# D9542 Fetal bovine serum Labtech Cat# FB-1001/100-500 Penicillin-Streptomycin Thermo Fisher Scientific Cat# 15070063 Trypsin-EDTA (0.25%), phenol red Thermo Fisher Scientific Cat# 25200056 HBSS, calcium/magnesium-free, no phenol red Thermo Fisher Scientific Cat# 14175095 DMEM Thermo Fisher Scientific Cat# 41965062 DMEM/F12 Thermo Fisher Scientific Cat# 11330032 Advanced DMEM/F12 Thermo Fisher Scientific Cat# 12634-010 Human EGF Thermo Fisher Scientific Cat# PHG0313 B-27 Supplement Thermo Fisher Scientific Cat# 17504044 Insulin solution human Sigma-Aldrich Cat# I9278 Insulin-Transferrin-Selenium (ITS) Thermo Fisher Scientific Cat# 41400045 GlutaMAX Supplement Thermo Fisher Scientific Cat# 35050061 Polybrene Sigma-Aldrich Cat# TR-1003-G Puromycin dihydrochloride APExBIO Cat# B7587 Antigenfix Diapath Cat# P0014 Zombie Aqua BioLegend Cat# 423101 ECL Chemiluminescence Detection Reagent Cytiva Cat# 10308449 D-luciferin PerkinElmer Cat# 122799 Critical commercial assays PowerUp SYBR Green Master Mix Thermo Fisher Scientific Cat# A25742 SuperScript IV VILO with ezDNase Thermo Fisher Scientific Cat# 11766050 LS columns Miltenyi Biotec Cat# 130-042-401 RNeasy Mini Kit QIAGEN Cat# 74904 RNeasy Micro Kit QIAGEN Cat# 74004 Deposited data RNA-seq This study GSE240040 ATAC-seq This study GSE240040 Experimental models: Cell lines 4T1 ATCC Cat# CRL-2539 CT26 ATCC Cat# CRL-2638 4T07 The Francis Crick Institute N/A TS1 Gift from J. Joyce17 N/A B16F10 ATCC Cat# CRL-6475 MLE12 ATCC Cat# CRL-2110 MLg ATCC Cat# CCL-206 Experimental models: Organisms/strains Mouse: FVB/n The Francis Crick Institute Jax Strain # 001800 Mouse: BALB/c The Francis Crick Institute Jax Strain # 000651 Mouse: Sftpc-CreERT2;R26REYFP C57BL/6 Gift from J-H. Lee N/A Mouse: Actin-GFP FVB/n Gift from J. Huelsken N/A Mouse: tdTomato C57BL/6 The Francis Crick Institute N/A Mouse: MMTV-PyMT FVB/n The Francis Crick Institute Jax Strain # 022974 Mouse: MMTV-PyMT C57BL/6 The Francis Crick Institute N/A Mouse: Actin-GFP MMTV-PyMT FVB/n The Francis Crick Institute N/A Mouse: TSLP KO FVB/n The Francis Crick Institute N/A (Continued on next page) e2 Developmental Cell 59, 1–16.e1–e8, September 23, 2024

Techniques: Ex Vivo, Generated, Control, Co-Culture Assay, Staining, Fluorescence

Journal: Developmental cell

Article Title: Bidirectional activation of stem-like programs between metastatic cancer and alveolar type 2 cells within the niche.

doi: 10.1016/j.devcel.2024.05.020

Figure Lengend Snippet: Figure 6. Cancer cell stemness governs alveolar epithelial cell reprogramming in the niche (A) Representative immunofluorescence image of 4T1 tumors growing in the primary site (mammary grand) or in the lung (metastasis). Tissues were stained with DAPI (blue), GFP (green), KRT8 (white), and Sox9 (red). Scale bars, 100 mm. (B) Quantification of the mean fluorescence intensity of nuclear Sox9 in cancer cells. Data are from one of two independent experiments, shown as mean ± SEM. (C) RT-qPCR for Sox9 in 4T1-GFP cancer cells isolated from primary tumor (PT) or lung metastasis (LM). Data are presented as mean ± SEM (n = 5 mice). (D) Quantification of the number of colonies generated from 4T1-GFP cells isolated from primary tumor (PT) or lung metastasis (LM). Data are presented as mean ± SEM (n = 3 mice). Each color identifies a different biological replicate. Representative images are indicated. (E) Schematics illustrating lung organoid assay in indirect co-culture with 4T1-GFP cells from the primary tumor (PT) or lung metastasis (LM). (F) Percentage of bronchioalveolar organoids formed. Data are presented as mean ± SEM of two independent experiments. (G) Western blot using cell lysates from 4T1 shControl or 4T1 shSox9 cancer cells with Sox9 and b-actin antibodies. Molecular weight (kDa) is indicated on the left. (H) Quantification of GFP+ cells in the lungs of BALB/c mice intravenously injected with 4T1-GFP cancer cells. Data are presented as mean ± SEM of two in- dependent experiments containing 3 mice each. (I) Weights of tumors formed upon mammary fat pad injection of 4T1-GFP cancer cells. Data are presented as mean ± SEM of two independent experiments containing 3–4 mice each. (J) Representative images of tumors from Figure 6I.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Deoxyribonuclease I from bovine pancreas Sigma-Aldrich Cat# DN25-100MG 4’,6-diamidino-2-phenylindole (DAPI) Sigma-Aldrich Cat# D9542 Fetal bovine serum Labtech Cat# FB-1001/100-500 Penicillin-Streptomycin Thermo Fisher Scientific Cat# 15070063 Trypsin-EDTA (0.25%), phenol red Thermo Fisher Scientific Cat# 25200056 HBSS, calcium/magnesium-free, no phenol red Thermo Fisher Scientific Cat# 14175095 DMEM Thermo Fisher Scientific Cat# 41965062 DMEM/F12 Thermo Fisher Scientific Cat# 11330032 Advanced DMEM/F12 Thermo Fisher Scientific Cat# 12634-010 Human EGF Thermo Fisher Scientific Cat# PHG0313 B-27 Supplement Thermo Fisher Scientific Cat# 17504044 Insulin solution human Sigma-Aldrich Cat# I9278 Insulin-Transferrin-Selenium (ITS) Thermo Fisher Scientific Cat# 41400045 GlutaMAX Supplement Thermo Fisher Scientific Cat# 35050061 Polybrene Sigma-Aldrich Cat# TR-1003-G Puromycin dihydrochloride APExBIO Cat# B7587 Antigenfix Diapath Cat# P0014 Zombie Aqua BioLegend Cat# 423101 ECL Chemiluminescence Detection Reagent Cytiva Cat# 10308449 D-luciferin PerkinElmer Cat# 122799 Critical commercial assays PowerUp SYBR Green Master Mix Thermo Fisher Scientific Cat# A25742 SuperScript IV VILO with ezDNase Thermo Fisher Scientific Cat# 11766050 LS columns Miltenyi Biotec Cat# 130-042-401 RNeasy Mini Kit QIAGEN Cat# 74904 RNeasy Micro Kit QIAGEN Cat# 74004 Deposited data RNA-seq This study GSE240040 ATAC-seq This study GSE240040 Experimental models: Cell lines 4T1 ATCC Cat# CRL-2539 CT26 ATCC Cat# CRL-2638 4T07 The Francis Crick Institute N/A TS1 Gift from J. Joyce17 N/A B16F10 ATCC Cat# CRL-6475 MLE12 ATCC Cat# CRL-2110 MLg ATCC Cat# CCL-206 Experimental models: Organisms/strains Mouse: FVB/n The Francis Crick Institute Jax Strain # 001800 Mouse: BALB/c The Francis Crick Institute Jax Strain # 000651 Mouse: Sftpc-CreERT2;R26REYFP C57BL/6 Gift from J-H. Lee N/A Mouse: Actin-GFP FVB/n Gift from J. Huelsken N/A Mouse: tdTomato C57BL/6 The Francis Crick Institute N/A Mouse: MMTV-PyMT FVB/n The Francis Crick Institute Jax Strain # 022974 Mouse: MMTV-PyMT C57BL/6 The Francis Crick Institute N/A Mouse: Actin-GFP MMTV-PyMT FVB/n The Francis Crick Institute N/A Mouse: TSLP KO FVB/n The Francis Crick Institute N/A (Continued on next page) e2 Developmental Cell 59, 1–16.e1–e8, September 23, 2024

Techniques: Staining, Quantitative RT-PCR, Isolation, Generated, Co-Culture Assay, Western Blot, Molecular Weight, Injection

Journal: Developmental cell

Article Title: Bidirectional activation of stem-like programs between metastatic cancer and alveolar type 2 cells within the niche.

doi: 10.1016/j.devcel.2024.05.020

Figure Lengend Snippet: Figure 7. Promotion of cancer stemness by AT2 cells ex vivo and in vivo (A) Schematics illustrating cancer organoid assay in co-culture with AT2 cells freshly isolated from tdTomato mice. (B) Fluorescence images GFP+ MMTV-PyMT tumoroids growing in the presence of AT2 cells (tdTom+) 7 days after plating. Scale bars, 100 mm. (C) Tumoroid-forming efficiency of MMTV-PyMT cells alone or in co-culture with AT2 cells. Data are presented as mean ± SEM (n = 4). (D) Experimental strategy and quantification of the tumoroid-forming efficiency of MMTV-PyMT cells growing with distal or niche lung epithelial cells. Each dot represents an independent experiment (n = 5). (E) Schematics illustrating in vivo tumorigenic assay of subcutaneous injections of 10,000 4T1 cancer cells alone or with 100,000 AT2 cells. (F) Bioluminescence quantification of luciferase-expressing 4T1 cells when injected alone or with AT2 cells subcutaneously (day 4). Each individual dot represents one mouse. (G) Bioluminescence imaging of mice injected with luciferase-expressing 4T1 cells alone (left) or with AT2 cells (right) subcutaneously. (H) Immunofluorescence image of 4T1 tumors stained with DAPI (blue), Sftpc (green), and KRT8 (magenta). Scale bars, 20 mm. (I) Fold change quantification of tumor weights at day 12 of 4T1 cells subcutaneously injected alone or with AT2 cells. (J) Representative images of the tumors as per Figure 7I. (K) Mini-list of the most upregulated genes (secreted) in niche lung epithelial cells versus distal. (L) Normalized counts of TSLP from bulk RNA-seq data. Each dot represents an independent biological replicate of 5 pooled lungs.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Deoxyribonuclease I from bovine pancreas Sigma-Aldrich Cat# DN25-100MG 4’,6-diamidino-2-phenylindole (DAPI) Sigma-Aldrich Cat# D9542 Fetal bovine serum Labtech Cat# FB-1001/100-500 Penicillin-Streptomycin Thermo Fisher Scientific Cat# 15070063 Trypsin-EDTA (0.25%), phenol red Thermo Fisher Scientific Cat# 25200056 HBSS, calcium/magnesium-free, no phenol red Thermo Fisher Scientific Cat# 14175095 DMEM Thermo Fisher Scientific Cat# 41965062 DMEM/F12 Thermo Fisher Scientific Cat# 11330032 Advanced DMEM/F12 Thermo Fisher Scientific Cat# 12634-010 Human EGF Thermo Fisher Scientific Cat# PHG0313 B-27 Supplement Thermo Fisher Scientific Cat# 17504044 Insulin solution human Sigma-Aldrich Cat# I9278 Insulin-Transferrin-Selenium (ITS) Thermo Fisher Scientific Cat# 41400045 GlutaMAX Supplement Thermo Fisher Scientific Cat# 35050061 Polybrene Sigma-Aldrich Cat# TR-1003-G Puromycin dihydrochloride APExBIO Cat# B7587 Antigenfix Diapath Cat# P0014 Zombie Aqua BioLegend Cat# 423101 ECL Chemiluminescence Detection Reagent Cytiva Cat# 10308449 D-luciferin PerkinElmer Cat# 122799 Critical commercial assays PowerUp SYBR Green Master Mix Thermo Fisher Scientific Cat# A25742 SuperScript IV VILO with ezDNase Thermo Fisher Scientific Cat# 11766050 LS columns Miltenyi Biotec Cat# 130-042-401 RNeasy Mini Kit QIAGEN Cat# 74904 RNeasy Micro Kit QIAGEN Cat# 74004 Deposited data RNA-seq This study GSE240040 ATAC-seq This study GSE240040 Experimental models: Cell lines 4T1 ATCC Cat# CRL-2539 CT26 ATCC Cat# CRL-2638 4T07 The Francis Crick Institute N/A TS1 Gift from J. Joyce17 N/A B16F10 ATCC Cat# CRL-6475 MLE12 ATCC Cat# CRL-2110 MLg ATCC Cat# CCL-206 Experimental models: Organisms/strains Mouse: FVB/n The Francis Crick Institute Jax Strain # 001800 Mouse: BALB/c The Francis Crick Institute Jax Strain # 000651 Mouse: Sftpc-CreERT2;R26REYFP C57BL/6 Gift from J-H. Lee N/A Mouse: Actin-GFP FVB/n Gift from J. Huelsken N/A Mouse: tdTomato C57BL/6 The Francis Crick Institute N/A Mouse: MMTV-PyMT FVB/n The Francis Crick Institute Jax Strain # 022974 Mouse: MMTV-PyMT C57BL/6 The Francis Crick Institute N/A Mouse: Actin-GFP MMTV-PyMT FVB/n The Francis Crick Institute N/A Mouse: TSLP KO FVB/n The Francis Crick Institute N/A (Continued on next page) e2 Developmental Cell 59, 1–16.e1–e8, September 23, 2024

Techniques: Ex Vivo, In Vivo, Co-Culture Assay, Isolation, Fluorescence, Luciferase, Expressing, Injection, Imaging, Staining, RNA Sequencing