Cancer-associated fibroblasts downregulate type I interferon receptor to stimulate intratumoral stromagenesis.

Activation of cancer-associated fibroblasts (CAFs) and ensuing desmoplasia play an important role in the growth and progression of solid tumors. Here we demonstrate that, within colon and pancreatic ductal adenocarcinoma tumors, efficient stromagenesis relies on downregulation of the IFNAR1 chain of the type I interferon (IFN1) receptor. Expression of the fibroblast activation protein (FAP) and accumulation of the extracellular matrix (ECM) was notably impaired in tumors grown in the Ifnar1S526A (SA) knock-in mice, which are deficient in IFNAR1 downregulation. Primary fibroblasts from these mice exhibited elevated levels of Smad7, a negative regulator of the transforming growth factor-β (TGFβ) pathway. Knockdown of Smad7 alleviated deficient ECM production in SA fibroblasts in response to TGFβ. Analysis of human colorectal cancers revealed an inverse correlation between IFNAR1 and FAP levels. Whereas growth of tumors in SA mice was stimulated by co-injection of wild type but not SA fibroblasts, genetic ablation of IFNAR1 in fibroblasts also accelerated tumor growth. We discuss how inactivation of IFNAR1 in CAFs acts to stimulate stromagenesis and tumor growth.

INTRODUCTION

Solid tumors grow in a complex and dynamic microenvironment populated by various types of cells [1]. Cancer-associated fibroblasts (CAFs) make up a large proportion of the cell population within the tumor microenvironment [2], and the presence of CAFs in the stroma has been associated with poor prognosis in many cancers, such as colorectal and pancreatic ductal adenocarcinomas (reviewed in [3, 4]). Protumorigenic activities of CAFs are mediated by a number of mechanisms, including secretion of growth factors and cytokines that promote malignant cell proliferation and immune evasion [5-8]. Furthermore, CAFs deposit and remodel the extracellular matrix (ECM) to generate a growth-supportive desmoplastic stroma [4, 9, 10]. Emerging evidence that support the pro-tumorigenic functions of CAFs have warranted the development and use of CAF-targeted therapies for cancer treatment [11-14] and prompted additional studies towards a better understanding of the molecular mechanisms driving the generation and activities of CAFs.

The stromagenic switch is the process in which resident and recruited fibroblasts are pathologically activated into CAFs [15]. A well-established inducer of CAFs activation is TGFβ [16, 17], whose signaling activity culminates in the upregulation of several CAF surface markers, including fibroblast activation protein (FAP) [18]. Although the TGFβ pathway is tightly regulated under physiological conditions, such as wound healing, it is dysregulated in the tumor microenvironment. While activated fibroblasts revert back to normal fibroblasts upon completion of wound healing, CAFs remain reprogrammed and pathologically activated throughout tumor progression [4, 9]. Therefore, it is likely, that under normal conditions, there exists a mechanism which acts to prevent the constitutive activation of fibroblasts. Moreover, this prohibitive mechanism is likely inactivated during tumor growth. Here we present data indicating that inactivation of the IFNAR1 chain of the Type I interferons (IFN1) receptor contributes to activation of CAFs and ensuing desmoplasia.

IFN1 are anti-tumor cytokines that signal and then elicit a massive reprogramming of transcriptional activities; all effects of IFN1 are mediated through the heterodimeric receptor complex composed of IFNAR1 and IFNAR2 chains [19, 20]. Tumors evade the IFN1 pathway by downregulating IFNAR1 in all types of cells within the tumor microenvironment, including intra-tumoral fibroblasts [21, 22]. Mice that express mutant IFNAR1 protein, which is deficient in its ubiquitination and, therefore, remain stable (Ifnar1, [23], henceforth termed “SA”) poorly support growth of solid tumors [22, 24]. Previously, we demonstrated that downregulation of IFNAR1 in the intra-tumoral cytotoxic T lymphocytes induces the formation of an immune-privileged niche in human colorectal cancers (CRC) and a mouse model of colon adenocarcinoma [22]. In the present study, we investigated the importance of IFNAR1 inactivation in the non-immune tumor stroma.

RESULTS

Within solid tumors, tumor microenvironment stress (e.g. inflammatory stimuli and hypoxia) downregulated IFNAR1 and inactivated the IFN1 pathway within all types of cells [22]. Downregulation of IFNAR1 in the host promoted growth of subcutaneous tumors formed by colon (MC38, Figure 1A) and pancreatic (MH6499c4, Figure 1B) adenocarcinoma cells as evident from delayed tumor growth in SA mice. Although inactivation of IFNAR1 in cytotoxic (CD8+) T cells plays an important role in this phenotype [22], we noticed that SA mice deficient in Rag1 and, accordingly, lacking lymphocytes, also exhibited decelerated tumor growth (Figure 1C). Furthermore, SA mice reconstituted with wild type (WT) bone marrow (WT>SA; Figure 1D) displayed limited tumor growth compared to WT chimeric recipients (WT>WT; Figure 1E). Collectively, these results suggest that downregulation of IFNAR1 in non-immune stromal cells is also important for robust tumor growth.

Figure 1.

Inactivation of IFNAR1 in the non-immune tumor stroma promotes tumor growth.

(A) . 106 MC38 cells were subcutaneously injected into the right flank of WT or SA mice (n=5 for each group). Tumors were measured by caliper and tumor area was calculated by multiplying the length and width. Each point in each line graph represents the mean ± S.D. tumor area (mm2). Statistical significance was determined by two-way ANOVA (**p<0.05, ***p<0.005, ****p<0.0001).

(B) 105 MH6499c4 cells were subcutaneously injected into the right flank of WT (n=4) or SA (n=5) mice. Each point in each line graph represents the mean ± S.D. tumor area (mm2). Statistical significance was determined by two-way ANOVA (**p<0.05, ***p<0.005).

(C) 106 MC38 cells were subcutaneously injected into the right flank of the indicated mice. Each point in each line graph represents the mean ± S.D. tumor area (mm2); n=5 for each experimental group. Tumors grown in WT mice were significantly larger than SA mice (*p<0.05, n=5), and tumors in Rag1−/−; SA were smaller than in Rag1−/−; WT mice (#p<0.05, n=5). Statistical significance was determined by two-way ANOVA.

(D) . Bone marrow cells were harvested from WT mice (donor). WT bone marrow cells were transplanted into irradiated WT (WT>WT) or SA mice (WT>SA) via tail-vein injection. 42 days post- transplantation, chimeras were inoculated s.c. with 106 MC38 cells.

(E) . Each point within each bar graph represents the tumor size (mm2) of a single mouse in each experimental group. Each bar represents the mean ± S.D in tumor size of four mice (n=4). Statistical significance was determined by student T-test (**p<0.05; ***p<0.005).

Examination of the stromal compartment in colon (Figure 2A) or pancreatic (Figure 2B) adenocarcinoma tumors revealed that tumors growing in SA hosts harbor developed limited stromagenesis. This phenotype was manifested by decreased levels of ECM components including fibronectin, versican and hyaluronic acid. Furthermore, the levels of FAP, an important marker of reactive/inflammatory CAFs [7] were lower in tumors growing in either SA mice (Figures 2A–B) or in chimeric SA mice transplanted with WT bone marrow (Figure 2C) indicating that intratumoral loss of IFNAR1 in the stroma promotes accumulation of CAFs. Together, these results suggest a role for IFNAR1 downregulation in the stromagenic switch and generation of pro-tumorigenic environment.

Figure 2.

Downregulation of IFNAR1 in fibroblasts induces the stromagenic switch.

(A-B) The expression levels of the ECM proteins Fibronectin (FN), Versican (VER), and Hyaluronic Acid (HA) in tumors was analyzed by IHC. Expression of the inflammatory CAFs marker FAP was evaluated by IF. The area of positive staining is presented as mean ± S.D. of at least 4 individual tumors (n=5 for MC38 CRC and n=4 for MH6499c4 PDA) with each dot representing a biological replicate. Fluorescence intensity is presented as mean ± S.D of 4 individual tumors (n=4), with each dot representing a biological replicate. Statistical significance was determined by Unpaired student t-tests (*p<0.05, **p<0.005, ***p<0.0005). All images were taken at 20X magnification. Scale bar = 50μm.

(C) The area of positive staining is presented as mean ± S.D. of 3 individual tumors (n=3) with each dot representing a biological replicate. Fluorescence intensity is presented as mean ± S.D of 3 individual tumors (n=3), with each dot representing a biological replicate. Statistical significance was determined by Unpaired student t-tests (*p<0.05, **p<0.005). All images were taken at 20X magnification. Scale bar = 50μm.

(D) WT and SA primary fibroblast-derived matrices were evaluated for their expression of FN and Smad7 by IF. Representative IF images are shown here. Fluorescence intensity of FN staining is presented as the mean ± S.D. of 6 independent experiments with each dot representing a biological replicate. Statistical significance was determined by Unpaired t tests (**p<0.005, n=6). The percentage of SMAD7-positive nuclei is presented as the mean ± S.D. of 3 independent experiments with each dot representing a biological replicate. Statistical significance was determined by Unpaired t-test (*p<0.05; n=3). All images were taken at 20X magnification. Scale bar = 50μm.

(E) NIH3T3 cells were serum-starved (0.1% BSA-DMEM) overnight, then pre-treated with 2000U/mL IFNβ for 2 hours and stimulated with 2.5 ng/ml TGFβ1 for an additional 4 hours. Cells were treated with PBS as a control. Whole cell lysates evaluated for expression of SMAD7 by Western blot analysis. GAPDH was used as a loading control. The numbers below each protein band indicates the integrated density of that band, with PBS-treated control set to 1. A representative blot is shown here (n=3).

(F) Matrices generated by SA lung fibroblasts infected with lentiviruses carrying either empty vector (CTRL) or shRNA against SMAD7 (shSMAD7) were evaluated for the expression of FN or Smad7 by IF. Fluorescence intensity of FN staining is shown as the mean ± S.D. of 3 independent experiments (n=3) with each dot representing a biological replicate. The percentage of SMAD7-positive nuclei is presented as the mean ± S.D. of 3 independent experiments (n=3) with each dot representing a biological replicate. Statistical significance was determined by Unpaired t-test (*p<0.05). All images were taken at 20X magnification. Scale bar = 50μm.

To test whether inactivation of IFN1 signaling is required for the stromagenic switch, we generated fibroblast-derived matrices (FDM) [25] using WT fibroblasts, in which IFNAR1 is readily degraded, or SA fibroblasts, in which IFNAR1 expression is preserved. While WT-FDM were abundant in fibronectin, SA-FDM were significantly less desmoplastic (Figure 2D). IFNγ was shown to induce a negative regulator of the TGFβ pathway, Smad7, in a manner that requires STAT1 [26], which is also activated by IFN1. Accordingly, attenuated production of fibronectin by SA fibroblasts coincided with increased levels of Smad7 (Figure 2D). Moreover, stimulation of IFN1 signaling in WT fibroblasts was sufficient to induce Smad7 expression (Figure 2E), while knockdown of Smad7 in SA fibroblasts restored their ability to generate a fibronectin-dense desmoplastic matrix (Figure 2F). These data suggest that inactivation of IFNAR1 in fibroblasts stimulates the stromagenic switch – at least in part through limiting Smad7 expression.

Importantly, in human CRC tumor tissues, the intra-tumoral stromal areas lacking IFNAR1 exhibited notably high expression of FAP (Figure 3A–C). Analysis of cells within the entire stromal compartment of human CRC showed almost mutually exclusive expression of FAP and IFNAR1 (Figure 3D–F). These results further suggest the importance of inactivation of IFNAR1 for the stromagenic switch and, given that low IFNAR1 [22] and high levels of FAP [27, 28] are associated with poor prognosis, argue for the importance of inactivation of IFNAR1 specifically in fibroblasts for supporting robust tumor growth.

Figure 3.

Inactivation of type I IFN signaling in CAFs promotes tumor growth.

Human CRC tumors (total of n=150) were evaluated for expression of IFNAR1 (red) and FAP (green) by IF. Malignant cells were stained with pan-cytokeratin (blue) and cell nuclei were stained with DAPI (teal). Scale bar=50μm; the inserts show lower magnification with larger tumor context. Quantification of FAP and IFNAR1 levels was completed using Tissue Studio image analysis software. User-guided machine learning was used for cell segmentation and identification of epithelial or stromal compartments of each tissue facilitated by DAPI-stained cell nuclei and pan-cytokeratin-stained cancer cells. Mean cell signal intensity of FAP and IFNAR1 was computed for epithelial and stromal regions. Scatterplots of single cell IFNAR1 and FAP signal intensities within the stromal compartment were generated per patient tumor core. Representative cases are shown that illustrate the generally mutual exclusivity of cellular FAP and IFNAR1 by predominantly FAP+/IFNAR1− tumors (Panels A and D), predominantly FAP−/IFNAR1+ (Panels B and E), or mixed FAP+/IFNAR1+ tumors (Panels C and E).

. Mice were inoculated with 105 MC38 cells alone (n=7), or with mixtures of 105 MC38 cells and 104 WT (n=9) or SA (n=5) fibroblasts. Each point in the line graphs represents the mean ± S.D tumor area (mm2). Statistical significance was determined by Two-way ANOVA and Sidak’s post-hoc test (****p<0.0001, n.s=not significant).

) . Col1a2-CreERT2/+ mice were crossed with Ifnar1fl/fl mice to generate Col1a2-CreERT2/+; Ifnar1fl/fl mice. Col1aI2-CreERT2/+; Ifnar1fl/fl mice were administered with tamoxifen (0.2 mg/g by oral gavage for 5 consecutive days). The resultant mice were called Ifnar1 mice.

) Genomic DNA from 4 mice were evaluated for expression of Col1a2-CreERT2/+ (top gel) and Ifnar1fl/fl (middle gel). A single band at 186bp indicated the presence of Col1a2-CreERT2/+. The floxed allele of Ifnar1 is represented by a band at 325, and the WT allele is represented by a band at 395bp. The presence of double bands at 325bp and 395 bp is indicative of a heterozygote. After tamoxifen treatment, mice were euthanized, and lung and ear fibroblasts were evaluated for Ifnar1 deletion by PCR (bottom gel). The absence of a band at 400bp indicates that Ifnar1 has been deleted. Thus, mice 1 and 2 are Ifnar1Δfib, while mice 3-5 are Ifnar1fl/fl.

Mice were inoculated with 105 MC38 cells. Each point in the line graphs represents the mean ± S.D tumor area (mm2) of 5 mice (n=5 for Ifnar1Δfib and Ifnar1fl/fl). Statistical significance was determined by Two-way ANOVA and Sidak’s post-hoc test (***p<0.005; ****p<0.0005).

The expression levels of FAP and SMAD7 in Ifnar1Δfib and Ifnar1fl/fl tumors were evaluated by IF. Fluorescence intensity is presented as mean ± S.D of 4 individual tumors (n=4), with each dot representing a biological replicate. Statistical significance was determined by Paired ratio t-tests (*p<0.05). All images were taken at 20X magnification. Scale bar = 50μm.

To test the role of downregulation of IFNAR1 in the pro-tumorigenic capacity of CAFs, we utilized two complementary approaches. First, we co-injected primary WT or SA fibroblasts along with MC38 tumor cells into SA mice. Supplementation of MC38 cells with WT (but not SA) fibroblasts accelerated tumor growth (Figure 3G). This result indicates that IFNAR1 downregulation within fibroblasts plays a role in establishing a pro-tumorigenic environment.

These results were corroborated by experiments involving a second independent approach, where we ablated IFNAR1 specifically in fibroblasts by tamoxifen treatment of Col1a2-CreER, Ifnar1fl/fl animals to generate Ifnar1Δfib mice (Figure 3H–I). Analysis of tumor growth in these animals revealed that ablation of IFNAR1 notably increased the size of tumors (Figure 3J). In these studies, tumors from Ifnar1Δfib mice exhibited a modest yet significant increase in FAP expression compared to tumors of comparable size grown for a longer time in Ifnar1fl/fl mice (Figure 3K). This increase is probably underestimated given that proteolytic downregulation of IFNAR1 in WT CAFs should blur the distinction with genetic ablation of IFNAR1. Altogether, these results suggest that inactivation of IFNAR1 in CAFs is required for their ability to support efficient tumor growth.

DISCUSSION

Our data indicate that proteolytic loss of IFNAR1 in the non-immune tumor microenvironment promoted stromagenesis and enhanced tumor growth, whereas stabilization of the receptor attenuated the pro-tumorigenic phenotype of CAFs. In human CRC tumors, IFNAR1 expression was spatially segregated from regions of high FAP expression, further demonstrating that loss of IFNAR1 is required for the pathological activation of CAFs. Altogether, these results suggest a novel mechanism by which the inactivation of the IFN1-IFNAR1 pathway enables the stromagenic switch and stimulates CAF-mediated tumor growth.

Inactivation of IFNAR1 and ensuing suppression of the IFN1 pathway in intra-tumoral cytotoxic T lymphocytes was shown to undermine their viability and drive the generation of an immune privileged niche within the tumor microenvironment [22]. In the present study, data obtained in experimental models that utilize bone marrow chimeras or animals lacking lymphocytes argue for the importance of IFNAR1 downregulation in the stromal compartment for tumor growth. The non-immune stromal compartment includes endothelial cells and fibroblasts. We noticed that angiogenesis was moderately inhibited in tumors grown in SA hosts (data not shown), and these results are consistent with importance of CAFs for activation of the endothelial cells [29] as well as with requirement of IFNAR1 downregulation for efficient VEGF-driven angiogenesis [30]. Thus, we cannot rule out additional importance of IFNAR1 inactivation in the endothelial cells in stimulation of tumor growth. However, based on experiments that involved IFNAR1 modulation specifically in fibroblasts, this study clearly points to the importance of downregulation of IFNAR1 in the activation and stromagenic function of CAFs. Future studies are warranted to examine the association between IFNAR1 levels in CAFs with immune infiltration of human tumors and potential responses to immunotherapies.

Results from our pre-clinical models of colon and pancreatic ductal adenocarcinoma tumors paralleled the data obtained in samples from human patients. Both showed that IFNAR1 expression in the tumor stroma was associated with low FAP expression. Considering that high FAP [27, 28] and low IFNAR1 [22] expression are associated with poor prognosis, our studies demonstrate the importance of IFNAR1 loss in intratumoral FAP+ inflammatory fibroblasts for their ability to support tumor progression.

Whereas our data implicate IFN1-induced expression of Smad7 as a potential mechanism mediating IFN1-driven suppression of stromagenesis, other putative mechanisms that could be inactivated within the tumor microenvironment cannot be ruled out. For example, downregulation of IFNAR1 promotes the uptake of the tumor-derived extracellular vesicles [31], which have been shown to stimulate activation of CAFs and ensued stromagenesis [32, 33].

The expression of the IL-1 receptor (IL1R1) is required for the pro-tumorigenic CAF phenotype [16], and IL-1 promotes downregulation of IFNAR1 in a manner that depends on activity of p38α protein kinase [34]. Intriguingly, recent studies of stromagenesis within pulmonary pre-metastatic niche suggested that p38-driven downregulation of IFNAR1 is essential for expression of FAP in the lungs of mice treated with the tumor-derived factors. Furthermore, these studies showed that decelerated tumor growth in mice lacking p38α in fibroblasts could be reverted by ablation of IFNAR1 [35].

Future studies will determine whether the blockade of the IL-1/IL1R1 and/or p38α pathways may stabilize IFNAR1 expression in fibroblasts and ultimately inhibit the stromagenic switch and CAFs-stimulated tumor growth. As desmoplasia promotes chemo-resistance in solid tumors; targeting the stromagenesis may improve the efficacy of anti-tumor therapies [36]. Accordingly, therapeutic modalities that stabilize IFNAR1 and re-activate the IFN1 pathway in fibroblasts may help to achieve this goal.

MATERIALS AND METHODS

Mouse Studies

All mouse experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of The University of Pennsylvania. Mice were maintained in a specific pathogen-free facility in accordance with the American Association for Laboratory Animal Science guidelines. All mice were of C57BL/6NJ background, and unless otherwise described are 6-8 weeks of age. Mice of both sexes were utilized for these studies. Rag1, and Col1a2-Cre mice were obtained from Jackson Laboratory (Bar Harbor, ME, USA). Ifnar1S526A mice (“SA”) and bone marrow chimeras were generated as previously described [23]. Fibroblast-specific IFNAR1 knockout mice (Ifnar1Δfib) were established by crossing Ifnar1fl/fl mice with Col1a2-CreERT2/+ mice. The resultant Col1a2-CreERT2/+; Ifnar1fl/fl mice were genotyped by PCR (Figure 3I). The Col1a2-CreERT2/+ was induced by administering tamoxifen (Sigma #T5648, St. Louis, MO, USA) 0.2 mg/g, daily for 5 days. Deletion of IFNAR1 was confirmed by PCR with primers for mouse wildtype IFNAR1 (Forward: 5’-CTGGGAGCCAGGGCATAAC-3’, Reverse 5’-CCAGCCTTTCGGAGTGTGC-3’) (Figure 3I). Littermates were randomly assigned into experimental groups, which were either co-housed or exposed to other groups’ bedding to ensure exposure to all groups’ microbiota.

Cell culture

MC38, NIH3T3 and HEK293T cells were maintained at 37°C with 5% CO2 in DMEM supplemented with 10% FBS, penicillin, and streptomycin. MH6499c4 mouse PDA cells were gifted by Dr. Ben Stanger from the University of Pennsylvania (Philadelphia) and maintained as previously described [37]. All cell lines were tested for mycoplasma contamination and were confirmed to be mycoplasma-free. For isolation of primary mouse fibroblasts, lungs harvested from 3- to 5-week-old mice were dissociated in PBS containing 100mg Collagenase II (Worthington #LS004202, Lakewood, NJ, USA) and 5mg DNAse I (Sigma #10104159001) and passed through 100 and 40 μm cell strainers to obtain single-cell suspensions. Fibroblasts were cultured for 20 minutes at 37°C, then non-adherent cells were washed off.

Immunohistochemical and immunofluorescent analysis of tumor tissues

OCT-embedded, frozen tumor tissues were cryosectioned in a Leica CM3050 S Cryostat at 10μm thickness for all immunohistochemical (IHC) and immunofluorescence (IF) studies. Slides were incubated overnight in primary antibody at 4°C. Secondary/tertiary antibodies were added and sections were developed and counterstained according to the appropriate protocol. For IHC, the following antibodies were used: anti-human fibronectin (Sigma #F3648), anti-mouse versican (EMD Millipore #AB1033, Burlington, MA, USA), biotinylated hyaluronic acid binding protein, HABP (EMD Millipore #385911), Rabbit IgG isotype control (Santa Cruz Biotechnology #sc-2027, Dallas, TX, USA), and biotin-SP-conjugated goat anti-rabbit IgG secondary antibody (Jackson Immunoresearch #AB2337959, West Grove, PA, USA). The following antibodies were used for IF: anti-human FAP antibody (R&D Systems #AF3715, Minneapolis, MN, USA or Abcam #53066, Cambridge, MA, USA) or sheep IgG isotype control (R&D Systems #5001A), and AF488-conjugated donkey anti-sheep IgG secondary antibody (ThermoFisher #A11015, Waltham, MA, USA). All imaging was completed with the Olympus Spot-RT3 microscope at 20X magnification. For quantification purposes, 3-5 different tumor samples (n≥3) from each experimental group were used. The mean area of positive staining and mean fluorescence intensity were determined using Fiji ImageJ Software.

Western Blot Analysis

Cells treated with IFNβ (PBL Assay Science #12400-1, Piscataway, NJ, USA) and recombinant mouse TGFβ1 (R&D systems #7666-MB) were lysed in RIPA buffer and whole cell lysates were evaluated for SMAD7 expression by Western blot analysis as described [23] using indicated antibodies (Smad7, ThermoFisher #42-0400 and GAPDH, Cell Signaling Technologies #14C10, Danvers, MA , USA). Densitometric analysis of the protein bands was completed with Fiji Image J.

FDM generation and analysis

Non-extracted (containing cells) FDMs were generated (as described in [25]) and analyzed for their expression of Fibronectin and Smad7 by immunofluorescence using antibodies against fibronectin (Sigma #F3648), Smad7 (ThermoFisher #42-0400), AF488-conjugated goat-anti-rabbit IgG (ThermoFisher #A11070) or AF594 conjugated anti-rabbit IgG (ThermoFisher #A11072). Imaging was completed with the Olympus Spot-RT3 microscope at 20X magnification. For quantification, 4-5 different fields of view of each type of matrix were used, and the matrix assembly assay was completed at least 3 independent times. The total area of positive FN staining and the % of SMAD7-positive nuclei was determined using ImageJ software. To calculate the % of SMAD7-positive nuclei, the following formula was utilized: (# of SMAD7 particles ÷ # of DAPI-stained nuclei) x 100%.

Lentiviral-mediated knockdown of SMAD7 in SA lung fibroblasts

pLKO-.1-shSMAD7 and pLKO.1-control plasmids [38] were gifted by Dr. Xin-Hua Feng (Zheijiang University, Hangzhou, China). For lentiviral production, 293T cells were transfected with pLKO-.1-shSMAD7 or pLKO.1-control plasmids followed by lentivirus harvest and transduction of SA fibroblasts as previously described [34].

Analysis of human cancers and statistical analysis

A human CRC tissue microarray (Cat# CRM1505) containing 150 cases of formalin-fixed, paraffin-embedded CRC cancer was obtained from US Biomax, Inc (Rockville, MD, USA). IF/IHC was performed as previously described [39] to detect IFNAR1 and FAP on a single slide using the following conditions: antigen retrieval with Tris/EDTA buffer pH 9.0, and incubation with rabbit polyclonal IFNAR1 antibody (Sigma, HPA018015), rabbit monoclonal FAP antibody (Abcam #ab207178), and mouse monoclonal anti-pan-cytokeratin (Agilent #M351501-2). Quantitative analysis was performed as previously described [39] using the Panoramic 250 Flash II slide scanner (3DHISTECH Ltd., Budapest, Hungary) to capture high-resolution digital images followed by quantification of biomarker levels using Tissue Studio image analysis software (Definiens, Munich, Germany). Mean biomarker signal intensity per cell was calculated for epithelial and stromal regions. Scatterplot of IFNAR1 and FAP signal intensities within each cell were generated per patient core. Representative cases shown.

Quantification and Statistical Analysis

All data are presented as mean ± s.e.m or mean ± s.d. (as indicated) of at least three biological replicates. Statistical analysis was performed using GraphPad Prism 8 software (GraphPad Prism Software Inc). Unpaired t-tests, or paired ratio t-tests were used for comparisons between two groups. One-way ANOVA or two-way ANOVA analyses followed by the Tukey post-hoc tests were used for multiple comparisons.