The CLCF1-CNTFR axis drives an immunosuppressive tumor microenvironment and blockade enhances the effects of established cancer therapies

Tumors comprise a complex ecosystem consisting of many cell types that communicate through secreted factors. Targeting these intercellular signaling networks remains an important challenge in cancer research. Cardiotrophin-like cytokine factor 1 (CLCF1) is an interleukin-6 (IL-6) family member secreted by cancer-associated fibroblasts (CAFs) that binds to ciliary neurotrophic factor receptor (CNTFR), promoting tumor growth in lung and liver cancer1,2. A high-affinity soluble receptor (eCNTFR-Fc) that sequesters CLCF1 has anti-oncogenic effects3. However, the role of CLCF1 in mediating cell-cell interactions in cancer has remained unclear. We demonstrate that eCNTFR–Fc has widespread effects on both tumor cells and the tumor microenvironment and can sensitize cancer cells to KRAS inhibitors or immune checkpoint blockade. After three weeks of treatment with eCNTFR-Fc, there is a shift from an immunosuppressive to an immunostimulatory macrophage phenotype as well as an increase in activated T, NKT, and NK cells. Combination of eCNTFR-Fc and αPD1 was significantly more effective than single-agent therapy in a syngeneic allograft model, and eCNTFR-Fc sensitizes tumor cells to αPD1 in a non-responsive GEM model of lung adenocarcinoma. These data suggest that combining eCNTFR-Fc with KRAS inhibition or with αPD1 is a novel therapeutic strategy for lung cancer and potentially other cancers in which these therapies have been used but to date with only modest effect. Overall, we demonstrate the potential of cancer therapies that target cytokines to alter the immune microenvironment.


Introduction
Lung cancer remains one of the most common cancers and there is a need for approaches to enhance currently available therapies.While inhibitors of KRAS G12C have shown e cacy in multiple clinical trials, resistance mechanisms rapidly emerge 4 .Similarly, while immune checkpoint inhibitors (ICIs) are effective in some patients, many do not respond 5,6 .The tumor microenvironment (TME) supports tumor growth via paracrine signaling from cancer-associated broblasts (CAF) 7 .Dysregulation of cytokines and other secreted factors is a universal feature in cancer, yet strategies to use this knowledge for therapeutic bene t have had limited impact 8 .Cytokine-directed therapies could be leveraged to enhance the effects of existing therapies by modulating the TME.CLCF1 (NNT-1/BSF-3) is a member of the IL-6 family of cytokines and is highly expressed in lung and pancreatic adenocarcinomas, as well as other tumor types (Extended Data Fig. 1) and was initially identi ed in T cell lymphomas 9 .The CLCF1 receptor, CNTFR, forms a trimeric complex with leukemia inhibitory factor receptor (LIFR) and gp130 10 .CLCF1 binding to this tripartite receptor complex triggers downstream signaling cascades mediated by JAK-STAT, MAPK, and other pathways 11 , driving tumorigenesis.We previously described a mechanism by which secretion of CLCF1 by CAFs promotes tumor growth in mouse models of lung adenocarcinoma 1 .eCNTFR-Fc is a soluble ligand trap that binds and sequesters CLCF1 3 .eCNTFR-Fc inhibits tumor growth as a single agent in xenograft and GEM models.Blockade of CLCF1 by eCNTFR-Fc has intrinsic effects on tumor cell signaling via STAT3 and Erk phosphorylation, leading to apoptosis and decreased proliferation in mouse tumor models 3 .Here, we dissect both the cell autonomous and non-cell autonomous effect of CLCF1 signaling in lung cancer using eCNTFR-Fc.Using single-cell and in situ spatial analysis, we demonstrate that CLCF1 plays a major role in driving an immunosuppressive TME and that combination of CLFCF1 inhibition with established cancer therapies can improve e cacy in NSCLC.These results establish a new therapeutic paradigm potentially widely relevant across many tumor types.

Results
Cell autonomous single agent eCNTFR-Fc treatment results in suppression of the RAS/MAPK signaling axis.
To assess the effect of eCNTFR-Fc on tumor development, we used an autochthonous, highly aggressive GEM model of lung adenocarcinoma 12,13 .Intratracheal instillation of adenovirus (Ad5-CMV-Cre) in Kras G12D−LSL /Trp53 f/f (KP) mice led to consistent development of adenocarcinoma.Tumors were induced in the lungs of KP mice and treated with vehicle or eCNTFR-Fc.Mice were sacri ced at one-or threeweeks after treatment initiation and lung cells harvested for analysis (Fig. 1a).To understand the effects of eCNTFR-Fc on tumorigenesis, treated tumors were analyzed using single-cell RNAseq and spatial transcriptomics.scRNAseq analysis was performed on 24,783 cells across all treatments.Cell types were identi ed based on reference cell annotations and manual curation (Fig. 1b and Extended Data Fig. 2a).Single cell transcriptome analysis of the tumor compartment identi ed effects of eCNTFR-Fc on genes related to immune modulation at 3 weeks (Fig. 1c).For example, eCNTFR-Fc upregulated Gst01, a gene coding for Glutathione transferase Omega 1, a protein previously shown to play a pro-in ammatory role 14 .eCNTFR-Fc also downregulated SelenBP1 expression which is associated with immune in ltration and is negatively correlated with the presence of NK cells, T helper cells, central and effector memory T cells and CD8 + T cells 15 .Pathway analysis showed enrichment of pathways related to immune function including antigen processing and presentation, viral infection and T cell receptor signaling (Fig. 1d).In contrast, the MAPK and PD-L1/PD1 immune checkpoint pathways were downregulated in tumor cells as shown by GSEA enrichment analysis (Fig. 1e-f and Extended Data Fig. 2b-c) at both 1-and 3-weeks post treatment.Downregulation of MAPK is consistent with prior data indicating that eCNTFR-Fc inhibits KRAS signaling 3 .PD-L1 signaling is possibly reduced due to decreased expression of Jun, Hif-1 a, and Nfkb in eCNTFR-Fc treated cells, all known regulators of PD-L1 expression 16,17 .Overall, eCNTFR-Fc treatment resulted in consistent dysregulation of 108 genes in the tumor cell compartment in the Kras G12D /Trp53 f/f model.
To further assess the consequences of eCNTFR-Fc on tumor cells, spatial analysis of changes in protein expression was performed on the Nanostring DSP Platform.Lung sections were stained for CD45 (yellow), PanCK (green), and DNA (blue) to establish overall tissue morphology.Regions of interest (ROIs) of 300 µm in diameter were selected for molecular pro ling with a 43-plex oligonucleotide-antibody cocktail designed to query the MAPK pathway and key immune subtypes (see methods).Examples of the lung morphologies are shown in Extended Data Fig. 3. Protein expression analysis in situ between vehicle and eCNTFR-Fc treated tumor cells also identi ed decreases in p42/44 MAPK and PD-L1 (Fig. 1g-h).
Differential protein expression analysis demonstrated decreases in total RAS, MEK, and ERK expression (Fig. 1i), which aligns with the decreases in gene expression seen using single cell transcriptome analysis.
RAS/MAPK pathway alteration in response to inhibition of the CLCF1/CNTFR signaling axis was also seen in human lung cancer cell lines (Extended Data Fig. 4).Human LUAD cell lines with stably integrated Cas9 were transfected with sgRNA guides to Control or CNTFR (Synthego) to generate Control (sgNT) or CNTFR knockout cell lines.Three KRAS mutant lines, (A549 (G12S), H23 (G12C), H358 (G12C)), and KRAS wildtype lines (H1437 and H1975) were evaluated.As previously reported, cell lines with KRAS mutations were generally more sensitive to KRAS knockout/knockdown 18 and KRAS mutant cell lines were more susceptible to eCNTFR treatment 3 .An exception was the KRAS G12C mutant cell line, H23, which did not show sensitivity to KRAS knockout (Extended Data Fig. 4a) consistent with previous reports [18][19][20][21] .The two most KRAS-dependent cell lines, determined by either knockout or with RNA interference, A549 and H358, were the most affected by CNTFR knockout (Extended Data Fig. 4b).Individual growth curves for A549, H358 and H23 with control or CNTFR knockouts conditions are shown in Extended Data Fig. 4c-e.Cells with a knockout of CNTFR had a lower capacity to form colonies compared to the non-targeting guides in A549 and H358, but not H23 (Extended Data Fig. 4f-h), consistent with the growth assay results.
To further de ne the tumor-intrinsic effects of CLCF1-CNTFR signaling, kinase signaling was analyzed under serum-starved and serum-stimulated conditions in sgNT and sgCNTFR A549 clones.CNTFR knockout resulted in abrogation of the JAK/STAT pathway and partial inhibition of the MEK/ERK and AKT pathways (Extended Data Fig. 5a).Western blotting also con rmed changes to the kinetics and intensity of ERK phosphorylation (Extended Data Fig. 5b-e).The effects on ERK signaling were not observed in H23 cells, which, as noted above, are refractory to KRAS inhibition (Extended Data Fig. 5f-g).In KRAS mutant (G12S) LUAD cell line A549, CNTFR knockout led to complete abrogation of the JAK/STAT and partial inhibition of the MEK/ERK and AKT pathways (Extended Data Fig. 5b-c).While H23 cells were not sensitive to either KRAS inhibition or CNTFR knockout alone, knockout of CNTFR increased sensitivity to KRAS G12C inhibition in H23 and H1792 knockout cell lines (Extended Data Fig. 6a-b).The same effect was observed when WT H23 cells were treated with eCNTFR-Fc in combination with KRAS G12C inhibition (Extended Data Fig. 6c).When KRAS G12C inhibition was combined with CNTFR knockout, there was an almost complete abrogation of p-ERK, indicating a synergistic effect on inhibition of MAPK signaling (Extended Data Fig. 6d-e).Overall, these results indicate that the CLCF1-CNTFR axis supports proliferation in the presence of KRAS inhibition and that abrogation of this axis using eCNTFR-Fc is a potential therapeutic strategy relevant to human cancers that carry KRAS mutations.
Single cell transcriptome analysis identi ed signi cant changes to immune cell populations in response to eCNTFR-Fc treatment.After one week of treatment with eCNTFR-Fc, macrophages were signi cantly depleted, whereas both B and T cells expanded (Fig. 2a).After three weeks of treatment, these changes were more apparent, with a strong enrichment of M1-like (Cd11c + Cd206 lo Cd86 hi ) macrophages and additional depletion of M2-like (Cd11c + Cd206 hi ) macrophages.At both treatment timepoints, there were fewer CD8 + T cells with exhaustion markers (Lag3, Ctla4, Pd-1, Tigit, and Havcr2) and an increase in cytotoxic CD8 + T cells, NKT cells, and NK cells.
The effect of eCNTFR-Fc treatment on T cells was further evaluated using differential gene expression analysis of both naïve and CD8 + subsets (Fig. 2b-c).After one week of eCNTFR-Fc treatment, there was a signi cant upregulation of ribosomal protein-coding genes (Fig. 2b), which is known to occur prior to T cell expansion 22 , consistent with the observed enrichment of mature effector T cell populations at the three-week timepoint.Differential gene expression analysis of eCNTFR-Fc treated CD8 + T cells and NKT cells at the three-week timepoint identi ed downregulation of these ribosomal proteins and upregulation of expression of Id2 and Gzma (Granzyme A) 22 , suggesting these cell populations have passed the expansion phase and have reached a mature cytotoxic phenotype (Fig. 2c).In parallel, MAPK signaling increased in T cells after eCNTFR-Fc treatment, in contrast to the observed effect in the tumor cell population and suggesting that this is an indirect effect.
Spatial analysis was used to analyze proteins associated with CLCF1-dependent changes in the immune microenvironment.After three weeks of treatment with eCNTFR-Fc, the T cell activation markers CD28 and MHC II were upregulated, whereas the T cell exhaustion markers CTLA4, PDL1, and PD-1 were downregulated 23,24 (Fig. 2e-f).CD28 is required for PD-1 blockade to e ciently kill cancer cells 25 .Ly6C/G expression was also decreased in CD45 + cells at both eCNTFR-Fc treatment timepoints (Fig. 2g).The Ly6 antibody has dual speci city, binding to both Ly6C and Ly6G, which marks two separate populations.
Ly6C marks a subset of monocytes/macrophages, and Ly6G marks neutrophils [26][27][28] , thus these two populations cannot be distinguished using this marker.The single cell analysis corroborates losses in both populations, with greater losses seen in the neutrophil populations when comparing vehicle to eCNTFR-Fc (Fig. 2a).Neutrophils have been shown to contribute to the immunosuppressive environment and facilitate immune evasion, reducing the effectiveness of immune checkpoint inhibition 29 .Taken together, eCNTFR-Fc treatment leads to an immune phenotype consistent with decreased immunosuppression and enrichment of activated effector immune cells.
Blockade of CLCF1 signaling potentiates the effect of checkpoint blockade.
The changes to the immune compartment after eCNTFR-Fc treatment suggest that the blockade of CLCF1 signaling could potentiate the effect of checkpoint inhibitor therapy in lung cancer.To test this, we tested the e cacy of combined therapy with eCNTFR-Fc and αPD-1 in the KP GEM model.At 8-weeks post tumor initiation, mice were treated with vehicle, eCNTFR-Fc alone, αPD-1 alone, or a combination of eCNTFR-Fc and αPD-1 for 28 days, and tumors were collected at 16 weeks (Fig. 3a).A representative image of lungs under each of the different treatments is shown in Fig. 3b.Mice treated with single agent eCNTFR-Fc demonstrated decreased tumor burden compared to vehicle-treated controls, while single agent αPD-1 had little effect (Fig. 3c).The effect of eCNTFR alone was less signi cant than in prior work 3 , most likely due to the higher tumor burden in mice treated in this study (see methods).Strikingly, combination of eCNTFR-Fc and αPD-1 led to a greater than additive effect and signi cantly decreased tumor burden when compared to vehicle, suggesting that eCNTFR-Fc is su cient to overcome the poor T cell responses, which usually renders immunotherapy ineffective in the KP lung adenocarcinoma model [30][31][32][33][34][35] .
To further dissect this drug interaction, we used a syngeneic allograft model where mouse LUAD cells, harvested from a GEM model mouse, were injected and grew subcutaneously in the mouse ank.Mice were implanted and treated with vehicle, eCNTFR-Fc alone, αPD-1 alone, or a combination of eCNTFR-Fc and αPD-1 for up to 28 days, and tumors were collected once mice reached endpoint (Fig. 3d).While eCNTFR-Fc or αPD-1 alone did have an effect on tumor growth in this model, with 7 of 18 tumors having decreased tumor volume and 2 achieving a complete response to eCNTFR-Fc alone and 4 of 19 tumors out of having decreased tumor volume and 3 achieving a complete response to αPD-1 alone.Mice that received a combination of eCNTFR-Fc and αPD-1 had signi cantly decreased tumor growth (Fig. 3e-f).11 of 22 mice had tumor regression, with 7 mice achieving a complete response, and the remaining mice exhibiting more intermediate responses (Fig. 3e-f).Combination treatment also signi cantly increased the survival of mice in comparison to the vehicle and single agent treated mice (Fig. 3g).
Composition of the immune system is altered after combination therapy with eCNTFR and αPD-1.
To evaluate the mechanistic basis for the enhanced response to αPD-1 when combined with eCNTFR-Fc, we performed single cell sequencing of lung tumors treated with single or combination therapy (Fig. 4a).
Treatment with αPD-1 alone resulted in the depletion of Cd206 hi M2-like macrophages and an increase of Cd86 hi Cd206 lo M1-like macrophages, similar to eCNTFR-Fc, but without the reciprocal enrichment of T cell effector populations.Cytotoxic NK, NKT, Cd8 + T cells and naïve T cells all were decreased after treatment with αPD-1.Notably, the only enrichment in T-cell populations after αPD-1 was an increase in exhausted CD8 + T cells, which can no longer mount an effective anti-tumor response.In contrast, when eCNTFR-Fc was combined with αPD-1, there was an overall decrease in the relative number of macrophages.As with single agent eCNTFR-Fc treatment, there was enrichment in cytotoxic CD8 + T cells, NKT cells, and NK cells, but additionally, there were increases in CD4 + T cells and T Regs (marked by Cd4 and Foxp3, respectively).Interestingly, T Regs have classically been shown to have decreased anti-tumor responses 36 ; however, some newer studies suggest careful control of CD4 expressing cells, including T Regs, are required for an optimal immune environment to promote anti-tumor e cacy of immunotherapy 37 .Subsetting of the macrophage population allowed for further characterization of these cells and increased resolution, allowing for the identi cation of 3 macrophage subpopulations (Fig. 4b): one M1-like (immunostimulatory) and two M2-like (immunosuppressive) populations.Treatment with eCNTFR-Fc resulted in the depletion of CD206 + M2-like macrophages, similar to single agent αPD-1 treatment (Fig. 4c).We observed expression of secreted cytokines in one subset of the M2-like macrophages.
Expression of a set of cytokines has been correlated to low response to immunotherapy in NSCLC 38,39 .Using this "refractory to PD-1" gene signature, we then calculated a score for the expression of the gene signature on a per-cell basis and identi ed an M2-like subset with a high PD-1 treatment refractory signature score (Fig. 4d), suggesting that these cells may partially mediate response to αPD-1.Additionally, it may suggest that eCNTFR-Fc contributes to the sensitization to αPD-1 by preventing the differentiation of naïve monocytes into this subset of macrophages, preventing subsequent secretion of refractory cytokines.
Although αPD-1 treatment alone is su cient to show the depletion of M2-like macrophages, this does not translate to the sensitivity to αPD-1 in the GEM model.This is likely due to the lack of reciprocal activation of effector cell populations and the possibility of secretion of refractory cytokines by multiple cell types (Extended Data Fig. 7), including other myeloid populations, monocytes, DCs and neutrophils, stromal cells, and tumor cells themselves.When looking at the response of tumor cells to treatment, tumor cells treated with αPD-1 alone showed increased expression of the genes associated with PD-1 refractory signature responses (Fig. 4e).However, treatment with eCNTFR-Fc alone or in combination with αPD-1 led to signi cant loss of the refractory signature, suggesting that suppression of the CLCF1/CNTFR signaling axis is preventing the release of these cytokines.This is not overly surprising as several genes in the signature are members of the IL6 subfamily or are targets of STAT3, which are canonically activated downstream components of CLCF1/CNTFR signaling 40,41 .To further con rm whether tumor cells are contributing to the cytokine expression changes in response to CLCF1/CNTFR signaling, we evaluated cytokine expression under serum-starved and serum-stimulated conditions in A549 control and CNTFR knockout cell lines using a cytokine array.CNTFR knockout led to decreased expression of IL-6 and IL-11 under both serum-starved and stimulated conditions (Extended Data Fig. 8).
VEGF 42 had decreased expression in CNTFR knockout cells and VEGF expression did not respond as well to serum stimulation, consistent with the observed decrease in STAT3 activation.VEGF has also been shown to directly in uence T cell response by increasing the expression of immunosuppressive checkpoints 43,44 .Similarly, we saw decreased expression of Angiopoietin-2, previously described as a biomarker of poor response to immunotherapy 45 and linked to poor prognosis for patients with NSCLC 46 .
To evaluate whether this response to combination treatment requires T cell activation, tumors were treated with a CD8 antibody to deplete CD8 + T cells in conjunction with the eCNTFR-Fc and αPD-1 combination therapy.CD8 + T cell depletion completely abolished the effect of the eCNTFR-Fc and αPD-1 combination (Fig. 4f-g), indicating that this effect is T-cell dependent.In summary, the combination of eCNTFR-Fc and αPD-1 treatment alters the cellular composition of the TME, leading to a phenotype of decreased immunosuppression and increased enrichment of effector immune cells and T cell mediated anti-tumor activity.

Discussion
We demonstrate a previously uncharacterized role for the CLCF1-CNTFR signaling in maintaining the protumorigenic TME of lung cancer.The blockade of this signaling pathway by eCNTFR-Fc has antioncogenic effects through two distinct mechanisms.First, cell lines and tumors sensitive to CNTFR knockout or inhibition show consistent changes to the KRAS/MAPK pathway.Additionally, KRAS mutant LUAD cell lines insensitive to KRAS inhibition become sensitized when CNTFR is knocked-out or treated with eCNTFR-Fc.These ndings support the hypothesis that CLCF1 potentiates oncogenic KRAS signaling in vivo and provides preclinical support for the use of eCNTFR-Fc in combination with the emerging class of direct KRAS inhibitors to treat KRAS mutant LUAD.
Single cell and spatial data indicate that eCNTFR-Fc broadly alters the TME from an immune-suppressive, tumor-promoting environment, towards a more immune-stimulatory, tumor-inhibitory phenotype.After treatment, we see decreased enrichment of immunosuppressive tumor-associated neutrophils and macrophages with increases in enrichment of effector T cells and NK cells.Combining eCNTFR-Fc with αPD-1 led to a signi cant increase in recruitment of cytotoxic NK, NKT and CD8 + T cells to tumors in a genetically engineered model of lung cancer that has been demonstrated to reproduce key features of the human disease.Treatment with eCNTFR-Fc sensitizes tumors to αPD-1 treatment in a highly aggressive GEM model, providing strong preclinical support for clinical trials of this combination.These results point to the potential increased e cacy of checkpoint inhibitor therapy when combined with agents such as eCNTFR that can reprogram the tumor microenvironment towards a more anti-oncogenic phenotype.
In hepatocellular carcinoma, CLCF1 affects the expression of CXCL6 and TGFb, promoting neutrophil recruitment and polarization of tumor-associated neutrophils towards the N2 phenotype 2 .These tumorassociated neutrophils, in turn, recruited tumor-associated macrophages, which promoted resistance to Sorafenib 47 .CLCF1 was also shown to contribute directly to Sorafenib resistance through PI3K/AKT signaling 48 .In glioblastoma (GBM), another IL6 family member, LIF, has also been shown to epigenetically silence CXCL9 in macrophages.Blockade of LIF removed silencing of CXCL9 expression, which promoted T cell migration and sensitization of GBM to αPD-1 therapy 49 .We previously established a LIFR decoy receptor for the treatment of pancreatic ductal adenocarcinoma 50 and, based on our results with eCNTFR-Fc, suggest targeting LIF in the context of αPD-1 combination therapy, which may also be of therapeutic bene t in LUAD.
Over the last several decades, there has been widespread interest in the idea of inhibiting cytokines and cytokine receptors as an anti-oncogenic strategy.Interleukins of the IL6 subfamily have been among the best studied 51 since the IL6-JAK-STAT signaling pathway is frequently altered in cancer and autoimmune diseases 52 .However, relatively little attention has been centered on the anti-oncogenic effects of CLCF1 speci cally.The results described here demonstrate the untapped potential of targeting the complex signaling networks between cells in the tumor microenvironment as a strategy to potentiate cancer therapy.
times weekly.Mice were euthanized at standard humane endpoints including when tumors reached 1000 mm 3 .Single Cell Experiment.Tracheas were exposed and the lungs were perfused with phosphate buffered saline (PBS).Whole lungs were removed, and tumors were macrodissected and minced with razor.Samples were then digested with Dulbecco's Modi ed Eagle Medium/F12 (no.11965-092, Gibco) containing 2 mg/mL collagenase/Dispase (no.11097113001, Roche Applied Science) and 0.025 mg/mL DNAse (no.AM2238, Invitrogen) at 37°C for one hour with agitation.Cells were ltered through 70 μm strainers.Red blood cells were lysed with hypotonic buffer (15mM NH4Cl, 10mM KHCO3 and 0.1mM EDTA) for one to two minutes, then washed with Dulbecco's Modi ed Eagle Medium/F12 supplemented with 10% fetal bovine serum (no.35-011-CV, Corning).Dead cells were removed using the MACS Dead Cell Removal Kit (no.130-090-101, Miltenyi Biotec) as per manufacturer's instructions.Cells were counted using a Countess II.17,000 cells were loaded onto the Chromium Next GEM Chip G. Library preparation was done as per 10x Genomics instructions and samples were sequenced on an Illumina NovaSeq 6000.
Transcripts were aligned to mouse (GRCh38) and a feature-barcode count matrix was generated using Cell Ranger (4.02).This was imported into R using the Seurat package (4.1.0)for downstream analysis 56 .Expression data was then tagged by experiment, scaled and cell-cell variation was corrected for by regressing out differences in number of detected molecules, cell cycle state 57,58 and percent mitochondrial gene content using the sctransform function 59 .Unsupervised principal component analysis, clustering, and non-linear dimensional reduction with UMAP was then used to detect the underlying cellular heterogeneity.Differential genes were identi ed using the FindAllMarkers function which uses a non-parameteric Wilcoxon rank sum test.Cluster identities were assigned through combination of immune ref using Single R and manual annotation.Differential gene expression analysis comparing two populations (ex.Vehicle vs eCNTFR) was performed using mixed modelling using MAST.The refractory to PD-1 gene list is shown in table 7 and refractory to PD-1 score was assessed by scoring each cell for depression of the signature using the AddModuleScore function.
Enrichment/depletion of cell clusters compared to vehicle treated tissue was determined using the Pearson residuals as per Pelka et al 60 .
NanoString.Formalin-xed, para n-embedded tissues were dewaxed in CitriSolv (no.1601, DeCon), then rehydrated in two washes each of 100% ethanol, 95% ethanol, and ddH20.Standard sodium citrate (pH 6.0) epitope retrieval was performed before washing in 1x TBS containing 0.1% Triton X-100 (no.TBST01-03, Bioland Chemicals).Samples were blocked in buffer W (no. 2-1003-100, Iba) for 1 hour at room temperature then incubated in primary antibodies overnight at 4 °C with UV-cleavable oligonucleotide-conjugated antibodies for DSP.The following day, nuclei were stained with a 1:10 dilution of SYTO 13 in 1x TBS-T for 15 minutes.Tissues were then incubated in 4% PFA for 30 minutes.Slides were then stained with the GeoMx solid tumor morphology kit (stains for DNA, PanCK and CD45) and oligo conjugated panels (Mouse Core Immune Cell Typing, Immune Cell Pro ling and MAPK Modules), tumors were selected as regions of interest (ROIs).ROIs were then segmented into tumor or immune cell populations based of Cd45 positivity and oligos collected for digital counts were obtained for each target on the nCounter system.CLCF1 patient expression.All TPM normalized counts for plotting gene expression are sourced from UCSC Treehouse (v11), representing a total of 12,499 samples from 49 different diseases.
Making Syngeneic Cell lines.Cell lines were generated from mice collected at the 16-week timepoint as above.Mouse lungs were harvested, and tumors were macrodissected.Cells were dissociated as above and then plated into 10 cm dishes.Cell lines were grown and then reimplanted to ensure histology matched lung adenocarcinoma.
Creation of CNTFR knockout cell lines.Human cell lines A549, H23, and H358 all were engineered to constitutively express Cas9 and then were transfected with sgRNA (Synthego).Brie y, 15 µL of 5 µM sgRNA in Opti-MEM (no.31985-070, Gibco) was added to Lipofectamine™ RNAiMAX Transfection Reagent (no.13778-150, Invitrogen) and incubated for 5 minutes.The transfection solution was then added to cells seeded the day prior.Media was changed after 24 hours.Single cell clones were created by performing a dual serial dilution starting with 10,000 cells.Colonies were allowed to form, then expanded and collected for veri cation of knockout.Knockout of CNTFR was veri ed using Inference of CRISPR Edits analysis (Synthego) on the sanger sequences.
Cell Assays.Cell Stimulations.When cells reached 70-80% con uence they were serum starved for 16 hours in serum-free RPMI-1640.Cells were then stimulated with recombinant human CLCF1 (10nM, no.962-CL, Biotechne) for 25 minutes.Cells were lysed and collected as previously described.
Cell Viability.Cells were seeded in 96-well plates at 500 cells per well in a total volume of 100μl of media containing 10% BGS.Plates were imaged by Incucyte (Sartorious) every 8 hours for 7 to 10 days.AMG510 Survival Curve.Cells were seeded in 96-well plates at 2,500 cells per well 24 hours before treatment.Cells were treated with increasing concentrations of AMG510.Plates were imaged by Incucyte (Sartorious) every 8 hours for 7 days.