Journal: bioRxiv

Article Title: A TERRA–NONO Axis Drives Fibroblast Reprogramming in Cancer

doi: 10.64898/2026.06.08.730872

Figure Lengend Snippet: (A) Chromosome-specific TERRA expression in multiple CAF strains isolated from skin SCC lesions (CAFs) compared to HDFs from adjacent unaffected skin (HDFs) from the same patients (#12, #13, #14, #16, #18, #19), as determined by RT-qPCR using specific primers. Expression is shown as fold change in CAFs versus matched HDFs, normalized to GAPDH. n = 6 (strains). Mean ± SD; two-tailed paired t-test. (B) RT-qPCR analysis of chromosome-specific TERRA expression in CAFs isolated from BCC lesions (BCAF6, BCAF7, BCAF8) and melanomas (MCAF508, MCAF603) compared to three age-matched reference HDFs (HDF11, HDF17, HDF18) normalized to GAPDH. n = 3 strains (BCAFs) n=2 strains (MCAFs). Mean ± SD. One-way ANOVA with Dunnett’s multiple comparisons test. (C) Representative images of TERRA FISH (red) and quantification of TERRA nuclear foci per cell, averaged per field, in CAFs compared to matched HDFs from the same patients. Nuclei were counterstained with DAPI (blue). RNase A treatment (100 µg/ml) was used as a negative control to assess probe specificity. n = 8 to 11 fields per sample. Mean ± SD. Unpaired t-test with Welch’s correction. Quantification of an additional CAF strain is shown in S1D. (D) Representative images and quantification of combined immunofluorescence (IF) and RNA FISH analysis of multiple SCC lesions compared to matched adjacent skin from the same patients. IF and RNA FISH were performed using a TERRA probe (red), anti-vimentin antibody (green), and DAPI (blue) for nuclear staining. Quantification represents the percentage of vimentin-positive cells with TERRA foci, averaged from four fields per patient. Images were acquired using a SoRA microscope. n = 5 patients. Mean ± SD. Paired t-test. (E) Representative images and quantification of combined immunofluorescence (IF) for vimentin, marking stromal cells, and RNA FISH using a TERRA probe in lung squamous cell carcinoma (SCC), lung adenocarcinoma, and normal lung tissues from a tissue microarray. Full-section vimentin staining is shown in S1F. Quantification indicates the percentage of vimentin-positive cells exhibiting TERRA foci per section. n = 3 normal tissues, n = 5 lung SCC, n = 8 lung adenocarcinoma. Mean ± SD. One-way ANOVA with Dunnett’s multiple comparisons test. (F) Representative images and quantification of combined immunofluorescence (IF) for vimentin, marking stromal cells, and RNA FISH using a TERRA probe in breast invasive ductal carcinoma, invasive lobular carcinoma, and normal breast tissues from a tissue microarray. Full-section vimentin staining is shown in S1G. Quantification indicates the percentage of vimentin-positive cells exhibiting TERRA foci per section. n = 3 normal tissues, n = 12 invasive ductal carcinoma, n = 2 invasive lobular carcinoma. Mean ± SD. One-way ANOVA with Dunnett’s multiple comparisons test.

Article Snippet: Additionally, HDFs and CAFs were treated with the NONO inhibitor (R)-SKBG-1 (MedChemExpress, Cat. No. HY-153918) at 5OμM for either 24 or 48 hours.

Techniques: Expressing, Isolation, Quantitative RT-PCR, Two Tailed Test, Negative Control, Immunofluorescence, Staining, Microscopy, Microarray

Journal: bioRxiv

Article Title: A TERRA–NONO Axis Drives Fibroblast Reprogramming in Cancer

doi: 10.64898/2026.06.08.730872

Figure Lengend Snippet: (A) Chromosome-specific TERRA expression in CAFs 48 hours after transfection with TERRA ASO or SCR ASO (100 nM). Data are expressed as relative fold change after GAPDH normalization. n=3 strains. Mean ± SD. Two-tailed paired t-test. (B) Representative images and quantification of TERRA nuclear foci per cell, averaged per field, detected by RNA FISH (red) in two strains of CAFs transfected with TERRA ASO versus SCR ASO control, as in the previous panel. Data represent pooled fields from 3 independent experiments (n=7 fields total). Mean ± SD. Unpaired t-test with Welch’s correction. (C) Representative images and quantification of TERRA nuclear foci per cell, averaged per field, detected by FISH (red) in HDFs infected with a TERRA overexpressing lentiviral vector (TERRA OE) versus a control vector (CTR). Data represent pooled fields from 3 independent experiments (n=10 fields total). Mean ± SD. Unpaired t-test with Welch’s correction. (D) Proximity ligation assays (PLA) with antibodies against TRF2 and g-H2AX in CAFs transfected with TERRA ASO or SCR ASO. Shown are representative images and quantification of PLA dots per cell per field. Data represent pooled fields from 3 independent experiments (n=12 fields total). Mean ± SD. Unpaired t-test with Welch’s correction. (E) Proximity ligation assays (PLA) with antibodies against TRF2 and γ-H2AX in HDFs upon infection with the TERRA overexpressing lentiviral vector (TERRA OE) versus control vector (CTR). Shown are representative images and quantification of PLA dots per cell per field. Data represent pooled fields from 3 independent experiments (n=10 fields total). Mean ± SD. Unpaired t-test with Welch’s correction. (F) Transcriptomic profiling of three CAF strains 48 hours after transfection with TERRA ASO or SCR ASO. Volcano plot displays differential gene expression between TERRA ASO and SCR ASO conditions. The x-axis shows the logO (fold change), and the y-axis shows the –logOO(p-value). Each dot represents a single gene; significantly differentially expressed genes are defined by p < 0.05 and an absolute logO (fold change) ≥ 0.58. Representative downregulated genes with known CAF effector functions are indicated. (G) Gene Set Enrichment Analysis (GSEA) of expression profiles from three CAF strains treated with TERRA ASO versus SCR ASO, analyzed against gene signatures associated with inflammatory CAFs (iCAFs), myofibroblastic CAFs (myCAFs), and genes upregulated in HDFs upon AR silencing . Genes were ranked by signal-to-noise ratio based on their differential expression between SCR ASO and TERRA ASO conditions. The positions of the genes in each set are indicated by black vertical bars, and the enrichment score is shown in green. Normalized enrichment scores (NES) and p-values indicate the degree and significance of enrichment or depletion in TERRA-silenced versus control CAFs. (H) Gene Set Enrichment Analysis (GSEA) of the indicated gene signatures from MSigDB. ( https://www.gsea-msigdb.org/gsea/msigdb/ ) including those of the CAF metaprogram CAF9 and CAF2 , and “Regulation of Inflammatory Response” (GO:0050727) and “Cytokine Production Involved in Inflammatory Response” (GO:0002534). The bar graph shows the - Log10 of p-value in profiles of control versus TERRA-silenced CAF. GSEA with 50 hallmark signatures is shown in Extended Data . (I) Expression of the indicated CAF effector genes in CAFs transfected with TERRA ASO versus SCR ASO assessed by RT-qPCR. Data are expressed as fold change relative to SCR ASO and normalized to GAPDH. n=3 CAF strains. Mean ± SD. Two-tailed paired t-test. (J) Expression of the indicated CAF effector genes in HDFs with TERRA overexpression compared to control, analyzed by RT-qPCR. Data expressed as fold change relative to CTR. Data normalized to RPLP0. n=3 HDF strains. Mean ± SD. Two-tailed paired t-test.

Article Snippet: Additionally, HDFs and CAFs were treated with the NONO inhibitor (R)-SKBG-1 (MedChemExpress, Cat. No. HY-153918) at 5OμM for either 24 or 48 hours.

Techniques: Expressing, Transfection, Two Tailed Test, Control, Infection, Plasmid Preparation, Ligation, Gene Expression, Quantitative Proteomics, Quantitative RT-PCR, Over Expression

Journal: bioRxiv

Article Title: A TERRA–NONO Axis Drives Fibroblast Reprogramming in Cancer

doi: 10.64898/2026.06.08.730872

Figure Lengend Snippet: (A) Expansion assays of SCC cells (SCC13) cocultured in a thin Matrigel layer with CAFs transfected with TERRA ASO versus SCR ASO controls (100nM). Shown are representative images of IF analysis with anti-pan-keratin (red) and vimentin (green) antibodies for SCC cells and CAFs identification, respectively, together with quantification of SCC cells expansion, measured by percentage of pan-keratin positive areas. Data represent pooled fields from 3 independent experiments (n=16 fields total). Mean ± SD. Unpaired t-test with Welch’s correction. (B) Expansion assays of SCC cells (SCC13) cocultured with HDFs infected with a TERRA overexpressing lentivirus (TERRA OE) versus control virus (CTR), utilizing the same conditions as in the previous panel. Shown are representative images and quantification of the results. Data represent pooled fields from 3 independent experiments (n=12 fields total). Mean ± SD. Unpaired t-test with Welch’s correction. (C) Intradermal tumorigenicity assays of SCC13 cells co-injected with CAFs transfected with either TERRA ASO or SCR ASO in contralateral mouse back skin. Shown are representative images of H&E-stained tumor sections along with quantification of tumor volume (mm³), calculated using the formula (Length × Width²) / 2 . n = 5 tumor pairs. mean ± SD. Two-tailed paired t-test. (D) Cancer cell density in lesions formed by SCC13 cells admixed with CAFs transfected with either TERRA ASO or SCR ASO, as in the previous panel. Shown are representative images of IF analysis with anti-pan keratin antibodies (red) and quantification of pan-keratin positive area, per lesion. n = 5 tumor pairs, 3 fields per sample. Mean±SD. Multiple unpaired t-test. (E) Intradermal tumorigenicity assays of SCC13 cells co-injected with HDFs infected with either a TERRA overexpression (TERRA OE) or control (CTR) in the contralateral mouse back skin. Shown are representative images of H&E-stained tumor sections along with quantification of tumor volume (mm³), calculated using the formula (Length × Width²) / 2 . n = 5 tumor pairs. mean ± SD. Two-tailed paired t-test. (F) Cancer cell density in lesions formed by SCC13 cells admixed with TERRA overexpressing HDFs versus controls as in the previous panel. Shown are representative images of IF analysis with anti-pan keratin antibodies (red) and quantification of pan-KRT positive area, per lesion. n = 5 tumor pairs, 3 fields per sample. Mean±SD. Multiple unpaired t-test.

Article Snippet: Additionally, HDFs and CAFs were treated with the NONO inhibitor (R)-SKBG-1 (MedChemExpress, Cat. No. HY-153918) at 5OμM for either 24 or 48 hours.

Techniques: Transfection, Infection, Control, Virus, Injection, Staining, Two Tailed Test, Over Expression

Journal: bioRxiv

Article Title: A TERRA–NONO Axis Drives Fibroblast Reprogramming in Cancer

doi: 10.64898/2026.06.08.730872

Figure Lengend Snippet: (A) Schematic representation of the iDRIP-MS (identification of direct RNA-interacting proteins – Mass Spec) workflow used to identify TERRA-interacting proteins in CAFs (Biorender). (B) Predicted interacting network of the identified TERRA-associated proteins based on experimental evidence, curated databases, or co-expression by STRING ( https://www.expasy.org/resources/string ), using a confidence score threshold of 0.4. The type of connections is denoted by lines of different colors. Known interactions (light blue, magenta), predicted interactions (green, red, blue), others (black, purple, lime). Note the NONO protein (in red) as one center of the network. (C) TERRA-NONO association in multiple CAF strains (CAF#19, CAF#24, CAF#26), as assessed by RNA immunoprecipitation (RIP) with antibodies against NONO versus non-immune IgGs, followed by RT-qPCR with primers specific to TERRA transcripts from chromosome 7p. Data are expressed as percentage of input. Mean±SD, paired two-tailed t -test. (D) PLA assay detecting endogenous AR/NONO interactions in CAFs versus matched HDFs. Red puncta indicate AR/NONO complexes; nuclei were counterstained with DAPI (blue). Quantification represents the number of PLA dots per cell per field. n = 5 fields per sample (counting > 20 cells per field), shown as mean ± SD. Unpaired t-test with Welch’s correction. (E) RIP assay performed using an antibody against NONO or control IgG in CAF or matched HDF (#27). TERRA levels from chromosome 7p were quantified by RT-qPCR and normalized to input. Data are presented as fold enrichment over control IgG. (F) NONO-AR association as detected by Proximity ligation assays (PLAs) of HDFs of HDFs plus/minus shRNA-mediated AR silencing as a control of specificity. Shown are representative images of nuclear NONO-AR complexes (red dots) together with quantification of PLA dots per cell per field. n = 5 fields (counting > 20 cells per field). Mean ± SD, one-way ANOVA with Dunnett’s multiple comparisons test. (G) RNA immunoprecipitation (RIP) was performed using an antibody against NONO or control IgG in HDFs transduced with control lentivirus (plko) or AR-targeting shRNAs (shAR57 and shAR18). TERRA levels from chromosome 7p were quantified by RT-qPCR and normalized to input. Data are presented as fold enrichment over control IgG. (H) PLA assays with antibodies against TRF2 and γ-H2AX in CAFs at 72 hours after transfection with NONO targeting siRNAs versus controls as in the previous panel. Shown arerepresentative images and quantification of the number of PLA puncta per cell per field. n =7 fields per sample (counting > 20 cells per field). Mean ± SD, one-way ANOVA with Dunnett’s multiple comparison correction. (I) Transcriptomic profiling of two CAF strains 72 hours after transfection with NONO targeting siRNAs versus controls. Shown is a volcano plot of differentially expressed genes with logOfold change (x-axis) and–logOOp-value (y-axis). Each dot represents a single gene; significantly differentially expressed genes are defined by p < 0.05 and an absolute logO (fold change) ≥ 0.58. Representative downregulated genes with known CAF effector functions are indicated. (J) Gene Set Enrichment Analysis (GSEA) plot using expression profiles from two CAF strains treated with siCTR or siNONO, analyzed against gene sets associated with inflammatory CAFs (iCAFs), myofibroblastic CAFs (myCAFs), and genes upregulated in HDFs upon AR silencing . Genes were ranked by signal-to-noise ratio based on their differential expression between siCTR and siNONO conditions. The positions of the genes in each set are indicated by black vertical bars, and the enrichment score is shown in green. Normalized enrichment scores (NES) and p-values indicate the degree and significance of enrichment or depletion in NONO-silenced CAFs.

Article Snippet: Additionally, HDFs and CAFs were treated with the NONO inhibitor (R)-SKBG-1 (MedChemExpress, Cat. No. HY-153918) at 5OμM for either 24 or 48 hours.

Techniques: Mass Spectrometry, Expressing, RNA Immunoprecipitation, Quantitative RT-PCR, Two Tailed Test, Control, Ligation, shRNA, Transduction, Transfection, Comparison, Quantitative Proteomics

Journal: bioRxiv

Article Title: A TERRA–NONO Axis Drives Fibroblast Reprogramming in Cancer

doi: 10.64898/2026.06.08.730872

Figure Lengend Snippet: (A) Expression of the indicated CAF effector genes in CAFs with DMSO or (R)-SKBG1 treatment (5 μM) after 24, 48 hours, analyzed by RT-qPCR. Data expressed as fold change relative to DMSO. Data normalized to RPLP0.n=3 biological replicates. Mean ± SD. One-way ANOVA with Dunnett’s multiple comparison test. (B) Expression of the indicated CAF effector genes in HDFs plus/minus AR silencing, followed by DMSO or (R)-SKBG1 treatment (5mM) for 48 hours analyzed by RT-qPCR. Data expressed as fold change relative to DMSO. Data normalized to RPLP0.n=3 biological replicates. Mean ± SD. One-way ANOVA with Šídák’s multiple comparisons test (C) RIP assay performed using an antibody against NONO or control IgG in CAF treated with DMSO or (R)-SKBG1 as described before. TERRA levels from chromosome 7p were quantified by RT-qPCR and normalized to input. Data are presented as fold of enrichment over control IgG. n=3 biological replicates. Mean ± SD. Paired two-tailed t-test. (D) Expansion assays of SCC cells (SCC13) alone or cocultured with CAFs treated with (R)-SKBG1 versus DMSO as above. Shown are representative images of immunofluorescence analysis with anti-pan-keratin (red) and anti-vimentin antibodies (green) for SCC13 and CAF identification, respectively, together with quantification of SCC cells expansion, measured by percentage of pan-keratin positive areas. Data represent pooled fields from 3 independent experiments (n>4 fields total). Mean ± SD. One-way ANOVA with Šídák’s multiple comparisons test. Note the significantly higher expansion of SCC cells admixed with CAFs relative to when tested in isolation, and the even greater suppression caused by (R)-SKBG1 treatment in the presence of CAF. (E) Spheroid invasion assays of SCC cells alone or admixed with CAFs and treated with DMSO or (R)-SKBG1 as described before. Shown are representative bright field images of spheroids and quantification of the invasion area measured as the difference between the core and the surrounding invaded area delimited with dotted lines. n >5 spheroids per condition. Mean ± SD. One-way ANOVA with Šídák’s multiple comparisons test. ns=0.3126 Note the significantly higher invasion capability of SCC cells admixed with CAFs relative to when tested in isolation, and the suppression by (R)-SKBG1 treatment. (F) SCC13 cells were co-injected intradermally with CAFs into the contralateral flanks of immunocompromised mice. Upon detection of palpable lesions (∼5 mm in diameter) 7 days post-injection, (R)-SKBG1 (10 µM diluted in PBS) or vehicle (PBS) was administered intratumorally into paired lesions once daily for 14 days. Shown are representative images of H&E-stained tumor sections and quantification of tumor volume (mm³), calculated using the formula (length × width²) / 2. n = 5 tumor pairs; data represent mean ± SD; statistical analysis was performed using a two-tailed paired t-test. (G) Cancer cell density in lesions formed by SCC13 cells admixed with CAFs treated with (R)-SKBG1 or PBS as in the previous panel. Shown are representative images of IF analysis with anti-pan keratin antibodies (red) to visualize SCC13 cells and with anti-vimentin (green) for SCC13 and CAF identification, respectively. Quantification of pan-keratin positive area, per lesion. n = 5 tumor pairs. Mean±SD, paired two-tailed t -test. (H) Representative immunofluorescence images of actinic keratosis (AK), squamous cell carcinoma (SCC), and lentigo maligna melanoma biopsies treated ex vivo with DMSO or the NONO inhibitor (R)-SKBG1 (10 µM) for 48 h. Sections were stained for periostin (POSTN; magenta), vimentin (Vim; green), keratin 5 (Krt5; yellow), and DAPI (blue). Lower panels: quantification of POSTN immunofluorescence signal intensity (integrated density) in AK, SCC, and melanoma lesions. Each point represents one field. Mean ± SD; Unpaired t test with Welch’s correction.

Article Snippet: Additionally, HDFs and CAFs were treated with the NONO inhibitor (R)-SKBG-1 (MedChemExpress, Cat. No. HY-153918) at 5OμM for either 24 or 48 hours.

Techniques: Expressing, Quantitative RT-PCR, Comparison, Control, Two Tailed Test, Immunofluorescence, Isolation, Injection, Staining, Ex Vivo