Synergy of chemotherapy and macrophage depletion leads to T cell memory activation and durable triple negative breast cancer regression
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE165987 | GEO | 2022/04/04
REPOSITORIES: GEO
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Project description:Purpose: Eliciting effective anti-tumor immune responses in patients who fail checkpoint inhibitor therapy is a critical challenge in cancer immunotherapy, and in such patients, tumor-associated myeloid cells and macrophages (TAMs) are promising therapeutic targets. We demonstrate in an autochthonous, poorly immunogenic mouse model of melanoma that combination therapy with an agonistic anti-CD40 mAb and CSF1R inhibitor potently suppressed tumor growth. Microwell assays to measure multiplex protein secretion by single cells identified that untreated tumors have distinct TAM subpopulations secreting MMP9 or co-secreting CCL17/22, characteristic of an M2-like state. Combination therapy reduced the frequency of these subsets, while simultaneously inducing a separate polyfunctional inflammatory TAM subset co-secreting TNF, IL-6, and IL-12. Tumor suppression by this combined therapy was partially dependent on T cells, TNF and IFN. Together, this study demonstrates the potential for targeting TAMs to convert a “cold” into an “inflamed” tumor microenvironment capable of eliciting protective T cell responses. Methods: Total RNA was purified with the use of QIAzol and RNeasy Mini kit (QIAGEN), in which an on-column DNase treatment was included. Purified RNA was submitted to the Yale Center for Genomic Analysis where it was subjected to mRNA isolation and library preparation. Non-strand specific libraries were generated from 50ng total RNA using the SMARTer Ultra Low Input RNA for Illumina Sequencing kit. Libraries were pooled, six samples per lane, and sequenced on an Illumina HiSeq 2500 (75-bp paired end reads), and aligned using STAR to the GRCm38 (mm10) reference genome. A count-based differential expression protocol was adapted for this analysis(Anders et al., 2013); mappable data were counted using HTSeq, and imported into R for differential expression analysis using the DESeq2.To find differentially regulated sets of genes for signature generation, a 1.5-Log2 fold-change difference between samples and p-adjusted (Holm-Sidak) ≤ 0.01 was used. Results: To begin to understand how these treatments modulated TAMs to control tumor growth, and to possibly illuminate additional biomarkers of response, we examined the transcriptomes of CD11b+ Ly6G- cells treated with CD40 or CSF1Ri, alone or in combination, relative to control, using high throughput RNA-sequencing. Principal components analysis (PCA) on the genome-wide dataset demonstrated that treating with CD40 and CSF1Ri individually caused largely non-overlapping changes in transcription, as indicated by their movement along orthogonal principal components (PC) relative to the control. Importantly, combination therapy was visualized as a systems-level combination of each individual treatment in PC space. We then examined the mRNAs most altered by either treatment alone or in combination relative to Controls (Log2FC>1.5, p<.01) by unsupervised hierarchical clustering. Five major gene patterns emerged from the clustering of genes. Cluster #1 comprises genes that are upregulated by CD40 and CSF1Ri+CD40 treatment but are mostly unaffected by CSF1Ri, suggesting that CD40 is the primary driver of this cluster in the combination treatment. Notable genes in this cluster include Tnfa, IfngIl12b and Cxcl9; interestingly, for Tnfa and Il12b, CSF1Ri+CD40 appears to have a synergistic effect on expression. In contrast to Cluster #1, Cluster #5 contains genes substantially downregulated by CSF1Ri and CSF1Ri+CD40 treatments, but are largely unaffected by CD40, suggesting that CSF1Ri is the driver of this cluster in the combination treatment. Cluster #5 genes include Cd36 and Fabp4, suggesting alterations in lipid homeostasis in the TAMs after treatment. Cluster #2 includes genes that are modestly upregulated by CD40 and CSF1Ri individually, leading to a stronger upregulation when combined. Finally, Clusters #3 and #4 include, for the most part, genes that are differentially affected by CD40 versus CSF1Ri and for which the combination treatment yields an intermediate response. In summary, these data show that CSF1Ri and CD40 agonism elicit predominantly distinct changes in gene expression in the CD11b+ cells, indicating they target different biological processes in myeloid cells. The net result of the changes in myeloid gene expression from the combination of CSF1Ri+CD40 treatment reveal additive effects by the individual treatments, but also synergy in the expression of several pro-inflammatory genes (e.g., Tnfa, Ifng, Il6 and Il12b). We further examined our dataset with Gene Set Enrichment Analysis (GSEA). Although CSF1Ri and CD40 treatments did not closely match any immunological signatures in the immunological database of MSigDb, combined CSF1Ri+CD40 had a strikingly similar signature to myeloid cells exposed to a variety of inflammatory stimulants, most closely reflected by BMDMs treated with lipopolysaccharide (LPS). This motivated us to look specifically at categories of NF-κB target genes that are significantly affected by LPS treatment, including transcription factors, cytokines and chemokines. Indeed, most of these NF-κB target genes associated with inflammation were strongly upregulated by CSF1Ri+CD40 treatment. Finally, Ingenuity Pathway Analysis identified TNFR1 and TNFR2 signaling and Acute phase response signaling among the top genetic signatures produced by the CSF1Ri+CD40 treatment combination, matching what we observed with GSEA. Thus, gene expression analysis not only revealed several biomarkers of response that may be relevant for assessing therapeutic activity in ongoing clinical trials using these drugs, but illuminated lead biological factors that may cause tumor regression. Conclusions: myeloid-targeted immunotherapies anti-CD40+CSF1R inhibition synergistically induce a pro-inflammatory microenviroment
2018-01-23 | GSE108753 | GEO
Project description:Purpose: Eliciting effective anti-tumor immune responses in patients who fail checkpoint inhibitor therapy is a critical challenge in cancer immunotherapy, and in such patients, tumor-associated myeloid cells and macrophages (TAMs) are promising therapeutic targets. We demonstrate in an autochthonous, poorly immunogenic mouse model of melanoma that combination therapy with an agonistic anti-CD40 mAb and CSF1R inhibitor potently suppressed tumor growth. Microwell assays to measure multiplex protein secretion by single cells identified that untreated tumors have distinct TAM subpopulations secreting MMP9 or co-secreting CCL17/22, characteristic of an M2-like state. Combination therapy reduced the frequency of these subsets, while simultaneously inducing a separate polyfunctional inflammatory TAM subset co-secreting TNF, IL-6, and IL-12. Tumor suppression by this combined therapy was partially dependent on T cells, TNF and IFN. Together, this study demonstrates the potential for targeting TAMs to convert a “cold” into an “inflamed” tumor microenvironment capable of eliciting protective T cell responses. Methods: Total RNA was purified with the use of QIAzol and RNeasy Mini kit (QIAGEN), in which an on-column DNase treatment was included. Purified RNA was submitted to the Yale Center for Genomic Analysis where it was subjected to mRNA isolation and library preparation. Non-strand specific libraries were generated from 50ng total RNA using the SMARTer Ultra Low Input RNA for Illumina Sequencing kit. Libraries were pooled, six samples per lane, and sequenced on an Illumina HiSeq 2500 (75-bp paired end reads), and aligned using STAR to the GRCm38 (mm10) reference genome. A count-based differential expression protocol was adapted for this analysis(Anders et al., 2013); mappable data were counted using HTSeq, and imported into R for differential expression analysis using the DESeq2.To find differentially regulated sets of genes for signature generation, a 1.5-Log2 fold-change difference between samples and p-adjusted (Holm-Sidak) ≤ 0.01 was used. Results: To begin to understand how these treatments modulated T cells to control tumor growth, and to possibly illuminate additional biomarkers of response, we examined the transcriptomes of CD11b+ Ly6G- cells treated with CD40 or CSF1Ri, alone or in combination, relative to control, using high throughput RNA-sequencing. Principal components analysis (PCA) on the genome-wide dataset demonstrated that treating with CD40 and CSF1Ri individually caused largely non-overlapping changes in transcription, as indicated by their movement along orthogonal principal components (PC) relative to the control. Importantly, combination therapy was visualized as a systems-level combination of each individual treatment in PC space. We then examined the mRNAs most altered by either treatment alone or in combination relative to Controls (Log2FC>1.5, p<.01) by unsupervised hierarchical clustering. Five major gene patterns emerged from the clustering of genes. Cluster #1 comprises genes that are upregulated by CD40 and CSF1Ri+CD40 treatment but are mostly unaffected by CSF1Ri, suggesting that CD40 is the primary driver of this cluster in the combination treatment. Notable genes in this cluster include Tnfa, IfngIl12b and Cxcl9; interestingly, for Tnfa and Il12b, CSF1Ri+CD40 appears to have a synergistic effect on expression. In contrast to Cluster #1, Cluster #5 contains genes substantially downregulated by CSF1Ri and CSF1Ri+CD40 treatments, but are largely unaffected by CD40, suggesting that CSF1Ri is the driver of this cluster in the combination treatment. Cluster #5 genes include Cd36 and Fabp4, suggesting alterations in lipid homeostasis in the TAMs after treatment. Cluster #2 includes genes that are modestly upregulated by CD40 and CSF1Ri individually, leading to a stronger upregulation when combined. Finally, Clusters #3 and #4 include, for the most part, genes that are differentially affected by CD40 versus CSF1Ri and for which the combination treatment yields an intermediate response. In summary, these data show that CSF1Ri and CD40 agonism elicit predominantly distinct changes in gene expression in the CD11b+ cells, indicating they target different biological processes in myeloid cells. The net result of the changes in myeloid gene expression from the combination of CSF1Ri+CD40 treatment reveal additive effects by the individual treatments, but also synergy in the expression of several pro-inflammatory genes (e.g., Tnfa, Ifng, Il6 and Il12b). We further examined our dataset with Gene Set Enrichment Analysis (GSEA). Although CSF1Ri and CD40 treatments did not closely match any immunological signatures in the immunological database of MSigDb, combined CSF1Ri+CD40 had a strikingly similar signature to myeloid cells exposed to a variety of inflammatory stimulants, most closely reflected by BMDMs treated with lipopolysaccharide (LPS). This motivated us to look specifically at categories of NF-κB target genes that are significantly affected by LPS treatment, including transcription factors, cytokines and chemokines. Indeed, most of these NF-κB target genes associated with inflammation were strongly upregulated by CSF1Ri+CD40 treatment. Finally, Ingenuity Pathway Analysis identified TNFR1 and TNFR2 signaling and Acute phase response signaling among the top genetic signatures produced by the CSF1Ri+CD40 treatment combination, matching what we observed with GSEA. Thus, gene expression analysis not only revealed several biomarkers of response that may be relevant for assessing therapeutic activity in ongoing clinical trials using these drugs, but illuminated lead biological factors that may cause tumor regression. Conclusions: myeloid-targeted immunotherapies anti-CD40+CSF1R inhibition synergistically induce a pro-inflammatory microenviroment
2017-12-27 | GSE108528 | GEO
Project description:Tumor associated macrophages (TAMs) are known to play a role in a multitude of processes that facilitate lung cancer growth, including the suppression of tumoricidal immune activation and the absorption of chemotherapeutics. Therefore, deciphering new therapies to reprogram TAMs towards an anti-tumor phenotype is at the cutting-edge of therapy development. The presence of iron-loaded macrophages has been associated with a better prognosis in lung cancer patients. Iron accumulation in macrophages stimulates pro-tumoricidal macrophage activity that triggers cytotoxic T-cell responses in the tumor microenvironment. In this study, we propose super-paramagnetic iron oxide nanoparticles (SPIONs) as a promising anti-lung cancer adjuvant therapy to reduce tumor cell growth. We used SPIONs to target iron to macrophages and observed that SPION-loaded macrophages reduced tumor cell growth due to the oxidative stress. Additionally, we found that SPIONs completely rewire the tumor microenvironment in mice towards an anti-tumor state and that in combination with crizotinib, a lung cancer targeted therapy, SPIONs show an additive effect in decelerating tumor growth
2024-04-18 | PXD040145 | Pride
Project description:Tumor-associated macrophages (TAMs), including anti-tumor M1-like TAMs and pro-tumor M2-like TAMs, are transcriptionally dynamic innate immune cells with diverse roles in lung cancer development. Epigenetic regulators are key controllers of macrophage fate in the spatially heterogeneous tumor microenvironment. Here, we demonstrate that the spatial proximity of histone deacetylase 2 (HDAC2) – overexpressing M2-like macrophages to tumor cells significantly correlates with poor overall survival of lung cancer patients. Suppression of HDAC2 in TAMs altered macrophage polarization, migration, and associated signaling pathways related to interleukin, chemokine, cytokine, and T cell activation. In various co-culture systems of TAMs and cancer cells, suppressing HDAC2 in TAMs results in reduced proliferation and migration and increased apoptosis of cancer cell lines and primary lung cancer cells, as well as attenuated endothelial cell tube length. HDAC2 regulates the pro-tumor macrophage phenotype via acetylation of histone H3 and transcription factor SP1. Myeloidcell–specificc deletion of Hdac2 (Hdac2f/fLysmCre) and pharmacological inhibition of class I HDACs in four different murine lung cancer models (intravenous, intratracheal, tumor relapse, and KrasLA2-driven) induced M2-like to M1-like macrophage phenotypic switching, altered infiltration of CD4 and CD8 T cells and retarded tumor growth and tumor microenvironmental angiogenesis. ThuTAM-specific HDAC2 expression may provide a novel strategy for lung cancer stratification and therapy.
2023-05-16 | GSE227560 | GEO
Project description:Hematopoietic stem/progenitor cells (HS/PCs) were transduced with lentiviral vectors overexpressing OFP and either miR-511-3p or a control, mutated miRNA sequence (miR-511-3p-mut). The transduced HS/PCs were then transplanted in recipient C57BL/6 mice. Tumors (Lewis lung carcinomas, LLC) were injected s.c. 4 weeks after HS/PC transplant. Lentiviral vector-transduced (OFP+), tumor-associated macrophages (TAMs) were isolated 4 weeks after LLC injection by fluorescence-activated cell sorting. Gene expression profiles of TAMs overexpressing either miR-511-3p or miR-511-3p-mut were obtained from 3 independent biological samples/each. Gene expression profiles of miRNA-overexpressing TAMs were then compared with the gene expression profiles of wild-type TAMs isolated from LLCs grown in nontransplanted C57BL/6 mice; the latter TAMs were subfractioned into MRC1+CD11c(low) or CD11c+MRC1(low) subsets before RNA isolation and analysis. Comparison of gene expression profiles of TAMs revealed that miR-511-3p overexpression tunes down the expression of multiple genes that are classically upregulated in protumoral MRC1+CD11c(low) TAMs. TAMs were isolated from Lewis lung carcinomas grown s.c. in C57BL/6 mice.
2012-04-01 | E-GEOD-34903 | biostudies-arrayexpress
Project description:Macrophages have been implicated in breast cancer progression and metastasis, but relatively little is known about the genes and pathways that are involved. Using a conditional allele of Ets2 in the mouse, we have identified Ets2 as a critical gene in tumor associated macrophages (TAMs) that specifically promotes mammary tumor metastasis. Loss of Ets2 in TAMs decreased the frequency and size of lung metastases without impacting primary tumor burden. Expression profiling of isolated tumor macrophages established that Ets2 deficiency resulted in the de-repression of a defined set of anti-angiogenic genes. Activation of this transcriptional program correlated with decreased angiogenesis in metastatic tumors and decreased metastatic growth. Comparison of this Ets2-specific TAM expression profile with human breast cancer profiles revealed a macrophage gene expression signature that could predict overall survival of estrogen receptor negative patients. In summary, we have identified a critical factor, Ets2, in TAMs that represses a transcriptional program to promote the growth of mammary tumor metastases in the lung. Breast TAMs were isolated from early-stage PyMT-induced mammary tumors expressing Ets2 and also from the tumors with Ets2-deficient TAMs. Since macrophages have also been implicated in normal mammary gland remodeling, normal remeodeling macrophages were also purified from females expressing Ets2 and the ones where Ets2 is deleted in the macrophages. One RNA sample was extracted from each genetic group for gene-expression profiling.
2010-01-05 | E-GEOD-12863 | biostudies-arrayexpress
Project description:In tumor microenvironment, tumor-associated macrophages (TAMs) have been characterized as M1-like or M2-like phenotype. In this study, we investigated the characteristics and functional roles of different TAMs on cancer metastasis. We isolated TAMs from primary tumor and metastatic lung and performed microarrays to identify the gene expression in distinct TAMs populations.
2018-07-26 | GSE96944 | GEO
Project description:C-C Motif Chemokine 18 (CCL18) belongs to the chemokine CC family and is predominantly secreted by M2-tumor associated macrophage (TAMs). It has been reported to be associated with various diseases and malignancies. Previous study showed that CCL18 promotes metastasis by activating downstream kinases. However, it remains unknown whether CCL18 regulates post-translational modifications, other than phosphorylation, during tumorigenesis. Here, we demonstrate that CCL18 is up-regulated in non-small cell lung cancer (NSCLC) and is involved in regulating the lysine acetylome in A549 cells. Using the combination of SILAC labeling and high-efficiency acetylation enrichment methods
2021-02-16 | PXD023468 | Pride
Project description:Tumor-associated macrophages (TAMs) have immunosuppressive capacity in mouse models of cancer. Here we show that the genetic deletion of the microRNA (miRNA)-processing enzyme DICER in TAMs broadly programs them to a CD11c+MRC1â/low M1-like immunostimulatory phenotype characterized by activated interferon-γ (IFN-γ)/STAT1/IRF signaling. M1-like TAM programming fostered the recruitment of cytotoxic T-cells (CTLs), including tumor-antigen-specific CTLs, inhibited tumor growth, and enhanced the efficacy of PD1 checkpoint blockade. Bioinformatics analysis of TAM transcriptomes identified a limited set of miRNAs putatively involved in TAM programming. Re-expression of Let-7 in Dicer-deficient TAMs was sufficient to partly rescue the M2-like (protumoral) TAM phenotype and abate tumor CTL infiltration. Targeted suppression of DICER activity in TAMs may, therefore, stimulate antitumor immunity and enhance the efficacy of cancer immunotherapy. To explore the role of DICER in the development, activation and immunological functions of TAMs, we crossed homozygous LysM-Cre (Clausen et al., 1999) with Dicerlox/lox (Harfe et al., 2005) mice to obtain mice with myeloid-cell-specific Dicer1 gene deletion (LysM-Cre;Dicerâ/â, referred to as Dâ/â). These mice were then backcrossed to LysM-Cre to obtain the control LysM-Cre; Dicer+/+ mice (referred to as D+/+). Both LysM-Cre and Dicerlox/lox mutations were always homozygous in our experiment. We then inoculated Lewis lung carcinoma (LLC) cells subcutaneously (s.c.) in Dâ/â and control D+/+ mice. Once the tumors were established, we isolated by fluorescence-activated cell sorting (FACS) tumor-associated macrophages (F4/80+ cells).
2016-06-13 | E-GEOD-76356 | biostudies-arrayexpress
Project description:PURPOSE: This tumor response pharmacodynamic model aims to describe primary lesion shrinkage in non-small cell lung cancer over time and determine if concentration-based exposure metrics for gemcitabine or that of its metabolites,n2',2'-difluorodeoxyuridine or gemcitabine triphosphate, are better than gemcitabine dose for prediction of individual response. EXPERIMENTAL DESIGN: Gemcitabine was given thrice weekly on days 1 and 8 in combination with carboplatin, which was given only on day 1 of every cycle. Gemcitabine amount in the body and area under the concentration-time curves of plasma gemcitabine, 2',2'-difluorodeoxyuridine, and intracellular gemcitabine triphosphate in white cells were compared to determine which best describes tumor shrinkage over time. Tumor growth kinetics were described using a Gompertz-like model.
RESULTS: The apparent half-life for the effect of gemcitabine was 7.67 weeks. The tumor turnover time constant was 21.8 week.cm. Baseline tumor size and gemcitabine amount in the body to attain 50% of tumor shrinkage were estimated to be 6.66 cm and 10,600 mg. There was no evidence of relapse during treatment.
2024-09-02 | BIOMD0000000234 | BioModels