Metabolic targeting of cancer associated fibroblasts overcomes T-cell exclusion and chemoresistance in soft-tissue sarcomas

T cell-based immunotherapies have exhibited promising outcomes in tumor control; however, their efficacy is limited in immune-excluded tumors. Cancer-associated fibroblasts (CAFs) play a pivotal role in shaping the tumor microenvironment and modulating immune infiltration. Despite the identification of distinct CAF subtypes using single-cell RNA-sequencing (scRNA-seq), their functional impact on hindering T-cell infiltration remains unclear, particularly in soft-tissue sarcomas (STS) characterized by low response rates to T cell-based therapies. In this study, we characterize the STS microenvironment using murine models (in female mice) with distinct immune composition by scRNA-seq, and identify a subset of CAFs we termed glycolytic cancer-associated fibroblasts (glyCAF). GlyCAF rely on GLUT1-dependent expression of CXCL16 to impede cytotoxic T-cell infiltration into the tumor parenchyma. Targeting glycolysis decreases T-cell restrictive glyCAF accumulation at the tumor margin, thereby enhancing T-cell infiltration and augmenting the efficacy of chemotherapy. These findings highlight avenues for combinatorial therapeutic interventions in sarcomas and possibly other solid tumors. Further investigations and clinical trials are needed to validate these potential strategies and translate them into clinical practice.


Statistics
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Software and code
Policy information about availability of computer code Data collection Flow Cytometry data were collected using the BD LRS II or Sony ID7000, cell sorting was performed with the BD Aria III.Western blot images were collected with the iBright imager (Thermo Fisher).RTqPCR data were generated with QuantStudio 3 (Applyed Biosystems).Single-cell partitioning was performed by using a 10X Chromium controller.RNA-seq data from TCGA Sarcoma PanCancer Atlas was queried in cBioPortal.H&E for the tumor sections were acquired by using a bright field microscope (Evos XL Core, Thermo Fisher).Immunofluorescence images were acquired using a fluorescence microscope (ECHO Revolve, ECHO).Seahorse Data was acquired using Agilent Seahorse XFp Analyzer.

Data analysis
All graphs were generated using Graphpad Prism v8 or R. T-test P-value was calculated either with Graphpad Prism v9.Flow Cytometry data were analyzed using FlowJo software v10.Multiplex immunofluorescence image analysis was performed in HALO HighPlexFL v3.6 (Indica Labs).Pathway enrichment analysis of RNA-seq data was performed with g:profiler.scRNAseq analyses used the following software: Cell Ranger (10X Genomics), Seurat, R v4.0, RStudio v1.3, ProjectTILs, and CellChat.Seahorse data was analyzed in Wave.
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Reporting on race, ethnicity, or other socially relevant groupings Not applicable.

Population characteristics
Not applicable.

Recruitment
Not applicable.

Ethics oversight
Human sarcomas for single-cell RNA sequencing were collected at Cedars Sinai Medical Center.Frozen tissue was provided by the Cedars Sinai BioBank and Research Pathology Resource, which received patient informed consent.FFPE blocks were provided by the Department of Pathology at Cedars Sinai.Because de-identified FFPE samples were used, this use does not qualify as human subject research, therefore no IRB approval was required.
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Life sciences study design
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Sample size
No statistical analysis was used to predetermine sample size.Sample size was chosen based on the standard protocols in the field.
All the independent biological replicates of the in vitro experiments are shown in the figures (single dots).For the mouse experiments, each mouse is shown as a dot in the presented charts.We used statistical analysis consistent with the sample size for each experiment and found sufficient statistical power with the sample sizes utilized in our study.
Data exclusions No data exclusion.

Replication
All studies, unless otherwise indicated, were performed at least three times with the exception of the transwell migration experiment which was performed twice.For in vivo experiments, multiple mice were used to ensure reproducibility.
Randomization In in vivo experiments animals were randomized to each experimental cohort.

Blinding
Tumor measurement and analysis was performed by operators blinded to the experimental groups.For the in vitro studies blinding was not relevant as all measures were quantified by standard cellular and biochemical assays.Key results were validated by 2 independent operators.
Reporting for specific materials, systems and methods

Wild animals
This study did not involve wild animals.

Reporting on sex
Syngeneic modeling of Ccne1+ and Vgll3+ tumors utilized mesenchymal stem cells isolated from female mice, thus sex-matched females were used as tumor recipients for all experiments.
Field-collected samples This study did not involve field collected samples.

Ethics oversight
Animal experiments were performed in accordance with the guidelines of Cedars-Sinai Medical Center Institutional Animal Care and Use Committee.
Note that full information on the approval of the study protocol must also be provided in the manuscript.

Flow Cytometry
Plots