Suppressing miR-21 activity in tumor-associated macrophages promotes an antitumor immune response

microRNA-21 (miR-21) is the most commonly upregulated miRNA in solid tumors. This cancer-associated microRNA (oncomiR) regulates various downstream effectors associated with tumor pathogenesis during all stages of carcinogenesis. In this study, we analyzed the function of miR-21 in noncancer cells of the tumor microenvironment to further evaluate its contribution to tumor progression. We report that the expression of miR-21 in cells of the tumor immune infiltrate, and in particular in macrophages, was responsible for promoting tumor growth. Absence of miR-21 expression in tumor- associated macrophages (TAMs), caused a global rewiring of their transcriptional regulatory network that was skewed toward a proinflammatory angiostatic phenotype. This promoted an antitumoral immune response characterized by a macrophage-mediated improvement of cytotoxic T-cell responses through the induction of cytokines and chemokines, including IL-12 and C-X-C motif chemokine 10. These effects translated to a reduction in tumor neovascularization and an induction of tumor cell death that led to decreased tumor growth. Additionally, using the carrier peptide pH (low) insertion peptide, we were able to target miR-21 in TAMs, which decreased tumor growth even under conditions where miR-21 expression was deficient in cancer cells. Consequently, miR-21 inhibition in TAMs induced an angiostatic and immunostimulatory activation with potential therapeutic implications.

Introduction miRNAs are small endogenous non-protein-coding RNAs that posttranscriptionally regulate the expression of multiple target genes in different cell types. In this manner, miRNAs control many physiological cellular processes, as well as several pathologies. Widespread dysregulation of miRNA expression has been reported in tumor cells, with either an oncogenic or a tumor suppressive role (1). Given their pivotal role in tumor initiation, progression, and response to therapy, these molecules have been accepted as potential cancer biomarkers (2). During neoplastic development, miRNAs have also been reported to be dysregulated in cells from the tumor microenvironment (TME) (3). Therefore, their role in cancer progression is not just limited to regulating cancer cell behavior. The nonmalignant cells of the TME have a dynamic and often tumor-supporting function (4), thus contributing to the clinicopathological profile and to the efficacy of anticancer therapy. The TME contains diverse types of cells, including cells of the immune system, endothelial cells, and fibroblasts. Recent studies have begun to unravel the cell-autonomous significance of miRNAs in macrophages, T cells, and endothelial cells (ECs) and their potential implications for cancer (5)(6)(7). miR-21 is the most commonly upregulated miRNA in solid tumors and hematological malignancies, and many studies have linked this cancer-associated microRNA (oncomiR) to poor prognosis and survival (8). The contribution of miR-21 to tumorigenesis has been extensively studied in the context of cancer cells, as it regulates various downstream effectors associated with tumor pathogenesis including cell invasion, proliferation, migration, apoptosis, and chemoresistance (8,9). The role of miR-21 in cells of the TME, however, has not been comprehensively studied. miR-21 is the most abundant miRNA in macrophages (10) and highly expressed in ECs and T lymphocytes (11,12). Several reports have described the role of miR-21 in regulating cellular functions that may have an impact on tumor growth, such as the inflammatory responses of macrophages, T-cell activation and endothelial angiogenic functions (12)(13)(14)(15)(16)(17)(18). However, the conclusions of these reports are inconsistent and in most cases are not directly studied microRNA-21 (miR-21) is the most commonly upregulated miRNA in solid tumors. This cancer-associated microRNA (oncomiR) regulates various downstream effectors associated with tumor pathogenesis during all stages of carcinogenesis. In this study, we analyzed the function of miR-21 in noncancer cells of the tumor microenvironment to further evaluate its contribution to tumor progression. We report that the expression of miR-21 in cells of the tumor immune infiltrate, and in particular in macrophages, was responsible for promoting tumor growth. Absence of miR-21 expression in tumor-associated macrophages (TAMs), caused a global rewiring of their transcriptional regulatory network that was skewed toward a proinflammatory angiostatic phenotype. This promoted an antitumoral immune response characterized by a macrophagemediated improvement of cytotoxic T-cell responses through the induction of cytokines and chemokines, including IL-12 and C-X-C motif chemokine 10. These effects translated to a reduction in tumor neovascularization and an induction of tumor cell death that led to decreased tumor growth. Additionally, using the carrier peptide pH (low) insertion peptide, we were able to target miR-21 in TAMs, which decreased tumor growth even under conditions where miR-21 expression was deficient in cancer cells. Consequently, miR-21 inhibition in TAMs induced an angiostatic and immunostimulatory activation with potential therapeutic implications.
exhibited an increased number of TUNEL-positive cells ( Figure  1C) and reduced tumor-associated vasculature, as shown by the diminished CD31 + vessel-like structures ( Figure 1D). These results indicate that loss of miR-21 increases tumor cell death, diminishes tumor angiogenesis, and provides evidence that miR-21 expression in cells other than cancer cells has an important role in promoting tumor growth.
Lack of miR-21 expression in immune cells is responsible for reduced tumor burden. To eliminate the role of stromal cells (e.g., fibroblasts, ECs) in limiting tumor growth of miR-21 -/mice, WT mice were lethally irradiated and subsequently transplanted with These results suggest that miR-21 expression within the tumor immune infiltrate is responsible for promoting tumor growth and that its deletion causes increased tumor cell death and decreased tumor angiogenesis.
Tumor immune infiltrate of miR-21 -/or WT mice adoptively transferred with miR-21 -/-BM is characterized by the presence of tumor-associated macrophages with an enhanced differentiated phenotype. Then, we examined the tumor-infiltrating immune cells in LLC tumors of either WT or miR-21 -/mice as well as WT mice adoptively transferred with WT or miR-21 -/-BM. We analyzed the frequency of immune cells related to tumor development, including myeloid-derived suppressor cells (MDSCs), tumor-infiltrating lymphocytes (TILs), and macrophages (4). We did not find differ-in the context of tumor progression. In other instances, the studies relied on culture systems to inhibit or overexpress miR-21, or approaches to globally target miR-21 in a non-cell/tissue-specific manner (19)(20)(21)(22)(23). Recent attempts to understand the role of miR-21 within immune infiltrating cells have also yielded conflicting results mostly due to lack of experimental data on cell-type specific KO or cell-type specific targeting approaches (24,25).
In this work, we have analyzed the function of miR-21 in noncancerous cells of the TME to determine its contribution to tumor progression. To this end, we used a syngeneic tumor mouse model and comprehensively assessed the effect of miR-21 deficiency in different cells/tissues on tumor progression. Briefly, our results indicate that the expression of miR-21 in macrophages of the tumor immune infiltrate plays a pivotal role in the regulation of tumor progression. Specifically, we found that both deletion and targeted inhibition of miR-21 in tumor-associated macrophages promotes proinflammatory angiostatic functions. This results in reduced tumor neovascularization and an antitumoral immune response, which is characterized by a macrophage-mediated improvement of cytotoxic T-cell responses. Together, these effects led to diminished tumor growth.
Results miR-21-deficient mice develop smaller tumors. In this study, we aimed to analyze the role of miR-21 in noncancerous cells of the TME on regulation of tumor progression. We used the Lewis lung carcinoma (LLC) model, which is a syngeneic, hence immunologically compatible model, and analyzed tumor growth in both WT and miR-21 KO mice (miR-21 -/-). As shown in Figure 1, A and B, miR-21 -/mice developed tumors with reduced volume and weight when compared with WT controls. Tumors of miR-21 -/mice  Figure 1, B and C). Furthermore, the proportion of IFNG expressing CD4 + or CD8 + T cells was similar in tumors from WT and miR-21 -/mice (Supplemental Figure 1D), suggesting that the percentage of activated T cells was equivalent. We did, however, find a significant reduction in TAM infiltration in miR-21 -/mice and mice transferred with miR-21 -/-BM ( Figure 3, A and B) and a decreased percentage of TAMs expressing C-C chemokine receptor type 2, although the level of surface expression was not affected (Supplemental Figure 1E). In both miR-21 -/animals and mice transferred with miR-21 -/-BM, we found that infiltrated TAMs exhibited increased surface expression of MHC class II (MHC II) (Figure 3, C and D). MHC II +high TAMs are better fosters of antitumor immune responses (26). Altogether, these data show that macrophages lacking miR-21 have reduced infiltration into the TME, and those present have a phenotype more associated with tumor growth suppression.
Expression of miR-21 in TAMs regulates disease outcome. miR-21 is the most consistently overexpressed miRNA across tumor types (8) and is associated with poor prognosis (27). In solid tumors, these analyses were based on total tumor RNA, which lacks critical spatial information. We used quantitative ISH (qISH) to directly assess the colocalization of miRNA expression with tumor epithelial and/or other subcellular compartments by multiplexing with DAPI and cytokeratin immunofluorescence (28,29). To determine the prognostic value of miR-21 expression in the major tumor compartments tumor islets [TIs] vs. TME), we used 3 retrospective non-small cell lung carcinoma (NSCLC) cohorts from Yale University (Supplemental Tables 1-3). NSCLC represents the most common subtype of lung cancer (30). These tissue microarrays (TMA) termed YTMA 79, 14, or 250, are composed of surgical resection samples from 290 patients with NSCLC. In order to validate miR-21 qISH, we first assessed miR-21 qISH on heart and ences in the percentage of MDSCs in the tumors of miR-21 -/mice or the proportion of monocytic or granulocytic MDSCs (Supplemental Figure 1A; supplemental material available online with this article; https://doi.org/10.1172/JCI127125DS1). We also did not detect differences in the percentage of CD4 + or CD8 + T cells in tumors from miR-21 -/mice or tumors from WT mice with miR-21 -/- with colorectal cancer (33). In patients with lung cancer, conflicting data have been reported (34,35). We used CD68 immunofluorescence to detect macrophages in our TMAs and found CD68 + macrophages present in all tumor TMA spots of NSCLC cohorts (n = 240) in both the TI and TME ( Figure 4D). A total of 50.1% of all miR-21-expressing cells were CD68 + ( Figure 4F). Although TME areas were slightly enriched in CD68 + miR-21 + cells ( Figure 4G), double positive cells could be found in both compartments ( Figure  4E), and the level of miR-21 expression was similar ( Figure 4E). We thus analyzed the contribution of miR-21 expression in macrophages to patients' survival. We found that the level of expression of miR-21 in CD68 + cells present within TI was not associated with 5-year OS ( Figure 4H). When the analysis was performed within the TME ( Figure 4I), however, there was significantly worse survival for patients with high levels of miR-21 in CD68 + cells when they were stratified by the median. Combined analysis of the expression of miR-21 in CD68 + cells of TI and the TME ( Figure  4J) showed a slight reduction in patients' survival when miR-21 is high in tumor macrophages. Altogether, these data indicate that although miR-21 expression in macrophages is heterogeneously localized within the tumor, the level of expression of miR-21 in macrophages and in particular, in macrophages within the TME can predict patient survival. miR-21-deficient TAMs express a gene signature associated with the promotion of a robust antitumor immune function. To elucidate the role of miR-21 in regulating TAM function, we isolated TAMs from LLC tumors of either miR-21 -/or WT mice and profiled their transcriptomes by RNA-Seq. miR-21 -/-TAM gene expression patterns were distinct from WT TAMs (Supplemental Figure 3A). Hierarchical clustering of log ratio transformed gene expression values of 1,035 genes that were differentially and significantly expressed (FC > 1.5, P ≤ 0.005 B-H FDR) showed the level of tran-spleen tissue from an miR-21 -/mouse (28). No specific signal for miR-21 could be detected in heart and spleen compared with normal WT (Supplemental Figure 2A). On the TMA, we found that the expression of miR-21 in tumor epithelial cells within TI was not associated with 5-year overall survival (OS) ( Figure 4A). In nontumor cells of the TME ( Figure 4B), however, there was significant prognostic effect on 5-year OS with a worse survival for patients with high levels of miR-21 expression if they were stratified by the median. Combined analysis of the expression of miR-21 in TI and the TME did not show a significant effect on OS ( Figure 4C). This finding indicates that high miR-21 expression in cells of the TME (non-tumor cells) has a negative impact on OS.
We then analyzed the expression of miR-21 in both compartments and found that the level of miR-21 expression was slightly higher in TI when compared with the TME ( Figure 4D). Despite this fact, high expression of miR-21 in the TME was associated with a worse prognostic value ( Figure 4B). This finding is in agreement with data showing that miR-21 expression in noncancerous cells of the tumor contributes to the pathogenesis of lung cancer (31). miR-21 is highly expressed in the monocytic/macrophage lineage (32) and the most abundant miRNA in macrophages (10). In order to determine the levels of miR-21 in immune cells of LLC tumors, we challenged WT mice with eGFP-LLCs and after 14 days, TAMs (CD45 + , CD11b + , MHC II + , F4/80 + ), LLCs (eGFP + ) and TILs (CD45 + , F4/80 -) were sorted and the miR-21 levels analyzed. Within the tumor, we found that miR-21 was highly expressed in TAMs compared with the rest of the immune infiltrate and tumor cells (eGFP-LLCs) (Supplemental Figure 2B). Likewise, miR-21 levels were higher in BM-derived macrophages (BMDMs) compared with LLCs in culture (Supplemental Figure 2C).
A high density of TAMs was associated with worse survival in gastric cancer and head and neck cancer but better OS in patients nication between innate and adaptive immune cells, Th1 pathway, and a proinflammatory gene profile ( Figure 5B). Conversely, a metabolic gene pattern, that in macrophages is linked to antiinflammatory functions, and a Th2 gene signature were diminished in miR-21 -/-TAMs ( Figure 5B). Similarly, the top overrepre-scriptional dysregulation between each genotype and highlighted the similar expression patterning found among biological replicates in each group ( Figure 5A). Ingenuity pathway analysis (IPA) of both up-and downregulated genes ( Figure 5B) in miR-21 -/-TAMs identified gene signatures that favor cytokine-mediated commu- sented disease and function categories pointed to an increased function of macrophages as antigen-presenting cells that promote activation of T lymphocytes ( Figure 5C). Alternatively, gene signatures related to tumor growth and the regulation of blood ves-sel formation were downregulated ( Figure 5C). Heatmaps with the list of regulated genes within these pathways and additional relevant functions are depicted in Figure 5, D-G and Supplemental Figure 3, B-D. Among the differentially expressed genes, Th1- promoting and tumor-suppressive cytokines such as Il27 and Il12 (36,37) and the costimulatory molecules (38), Cd86 and Cd40, were upregulated in miR-21 -/-TAMs (Supplemental Figure 3E). Moreover, we found increased expression of proinflammatory mediators including Tnf and Cxcl10 and Cxcl9 (Supplemental Figure 3E). IL-12 is a reported target of mir-21 (15) and a strong inducer of Th1 responses, with demonstrated antitumor activity (37) that is produced by different immune cells including macrophages. The increased levels of Il12 found in miR-21 -/-TAMs are consistent with lost miR-21-mediated targeting. To identify miR-21/mRNA associations relevant to TAM pathophysiology, we examined global correlation patterns between significantly upregulated genes in miR-21 -/-TAMs and miR-21 predicted target genes (Figure 6A). We used a combination of 4 microRNA prediction algorithms and selected genes that were predicted by at least 2 prediction methods. Among the 321 significantly upregulated mRNAs, 65 were predicted targets of miR-21 ( Figure 6A). Together, they represented approximately 20% of the significantly upregulated genes found in miR-21 -/-TAMs. These 65 significantly upregulated and predicted targets of miR-21 were used as a data set for network analysis using the IPA Regulator Effects algorithm (Figure 6B). Downregulation of miR-21 was predicted as an upstream regulator, providing confidence of the predicted regulatory network generated. Among the predicted upstream regulators, we found IL-21, IFNA, and IFNG, factors reported to have antitumor effects in a variety of murine experimental tumor models (39)(40)(41). The model also predicts IL-1B, a canonical proinflammatory cytokine with antitumor roles (42), as an upstream regulator. The anticipated impact of the molecular expression changes showed an increase in downstream functions involved in inflammatory responses as well as the activation and homeostasis of leukocytes. These data indicate that miR-21 depletion in TAMs causes a global rewiring of their transcriptional regulatory network that is skewed toward a proinflammatory phenotype and the promotion of an antitumoral immune response (5,42,43). Proinflammatory stimulation of miR-21 -/-BMDMs revealed a significant upregulation of proinflammatory gene signature, including Il12, Tnf, and Cxcl10 compared with WT BMDMs (Supplemental Figure 3F). These findings are in agreement with previous published data (10,16) and support the idea that miR-21 deficiency primes macrophages to develop a proinflammatory program (43). In vivo, this translates to an antitumor phenotype when it happens in conjunction with Th1 immune response (5,42). Interestingly, a proinflammatory phenotype of TAMs in patients with NSCLC has been positively associated with survival time (44).
Increased IL12 in miR-21-deficient TAMs improves cytotoxicity of CD8 + T cells. Consistent with the TAM profiling data, we found that tumors from miR-21 -/mice had a higher percentage of IL-12 + and TNF + TAMs (Figure 7, A and B, left panels) and also had higher levels of IL-12 and TNF (Figure 7, A and B, right panels). Similar results were observed in the tumors of WT mice transplanted with miR-21 -/-BM (Figure 7, C and D). We also found increased levels of costimulatory molecules CD40 and CD86 ( Figure 7E) linked to enhanced macrophage antitumor activity (38). These data, together with the overall transcriptome signature exhibited by miR-21 -/-TAMs, suggest that these cells could participate in enhancement of cytotoxic T-lymphocyte (CTL)-dependent cell death (45)(46)(47).
Increased intracellular levels of GZMB and higher levels of extracellular LAMP1 (CD107a) in tumor-infiltrated CD8 + T cells indicate better targeting and killing capacity of cancer cells by CTLs (48). We found that CD8 + T cells within the tumors of mice transplanted with miR-21 -/-BM had higher levels of GZMB than their WT counterparts (Figure 7, F and G) and enhanced degranulation status (Figure 7, H and I), as indicated by the larger number of CD8 + T cells with surface expression of CD107a (48). This finding is in agreement with the increased cell death observed in tumors of miR-21 -/or WT mice transplanted with miR-21 -/-BM. However, the phenotype observed in CD8 + TILs could be mediated by the absence of miR-21 expression in these cells. Thus, we analyzed GZMB levels in CD8 + T cells isolated from the Peyer's patches of healthy adult WT or miR-21 -/mice because in healthy and unchallenged mice, CD8 + T cells from Peyer's patches express high levels of GZMB (49). We did not find differences in GZMB levels (Supplemental Figure 3G). Additionally, naïve splenic CD8 + T cells of miR-21 -/mice activated by anti-CD3 Ab for 3 and 6 days exhibited no difference in intracellular levels of GZMB (Supplemental Figure 3H) compared with WT controls. These results indicate that the absence of miR-21 in CD8 + T cells does not affect cell-intrinsic levels of GZMB and that additional mechanisms account for the observed differences in CD8 + TILs from miR-21 -/mice.
To test whether the increase of GZMB and CD107a in CD8 + TILs of miR-21 -/mice leads to more CTL activity, we isolated CD8 + TILs from WT or miR-21 -/tumors and immediately cultured them with LLCs to monitor their killing capacity. CD8 + TILs isolated from miR-21 -/mice were more effective at reducing proliferation and increasing the number of apoptotic (annexin V + ) target cells (Figure 7, J and K), indicating that miR-21 deficiency in non-CD8 + cells within the TME improves CTL activity.
IL-12, a potent inducer of CTL activity in CD8 + T cells (37), was increased in TAMs of miR-21 -/mice or WT mice transplanted with miR-21 -/-BM (Figure 7, A and C). Thus, we hypothesized that increased IL-12 in miR-21 -/-TAMs was responsible for the increased GZMB levels. Thereby, WT splenic CD8 + T cells were isolated and activated with an anti-CD3 Ab and incubated with conditioned media obtained from miR-21 -/or WT TAMs ( Figure  7L). Conditioned media from miR-21 -/-TAMs led to higher levels of GZMB than media obtained from WT TAMs. Furthermore, a neutralizing anti-IL-12 Ab blunted this effect ( Figure 7L). Altogether, these data suggest that IL-12 from miR-21 -/-TAMs participates in the improved T-cell responses observed in mice transferred with miR-21 -/-BM and thus contributes to the increased tumor cell death observed in these animals, consistent with the enhanced antitumor immune response mediated by miR-21 -/-TAMs.
Increased CXCL10 in miR-21-deficient TAMs mediates angiostatic effects within the LLC tumors. Reduced vascularization was another outstanding phenotype observed in tumors of mice transplanted with miR-21 -/-BM. mir-21 -/-TAMs exhibited a gene signature associated with decreased blood vessel formation. In addition to Il12, miR-21 -/-TAMs exhibited increased mRNA levels of Cxcl10 and Cxcl9. IL-12 in tumors negatively regulates the tumor vasculature (50). This effect is associated with IFN-inducible production of CXCL10 and CXCL9, angiostatic chemokines that have been described to halt tumor progression by inhibiting EC proliferation and differentiation into capillary structures (51). Immunofluorescent staining jci.org Volume 129 Number 12 December 2019 production of IL-12 and CXCL10 in TAMs. This can explain, at least in part, the increased tumor cell death and reduced neovascularization, leading to an overall reduction of tumor growth. Conditional deletion of miR-21 in macrophages reduces tumor growth by promoting CTL activity and diminishing the proliferative phenotype of tumor-associated vasculature via increased IL-12 and demonstrated that CXCL10 was significantly increased in CD68 + TAMs from WT mice transplanted with miR-21 -/-BM ( Figure 8A). Moreover, tumor ECs (TECs) isolated from these mice showed reduced numbers of Ki-67 + cells compared with their control counterparts ( Figure 8B), indicating diminished proliferation. These results indicate that absence of miR-21 induces the expression and  To better understand the interaction of miR-21-deficient macrophages with CD8 + T cells in the context of the TME, we performed single-cell RNA-Seq in sorted CD45 + cells from the tumors of miR-21 fl/fl or LysMCre;miR-21 fl/fl mice. We first used unsupervised clustering to generate t-distributed stochastic neighbor embedding (t-SNE) plots that separated the cells into distinct groups ( Figure 11, A and B). Representation of t-SNE plots (Figure 11, overlay colored by sample) did not reveal overall outstanding differences in cell populations. The same colored cells were clustered together in the combined sample ( Figure 11B). We identified and classified 7 major cell types: CD8 + T cells, CD4 + T cells, NK cells, granulocytic MDSCs, monocytic MDSCs, DCs and macrophages/ monocytes (see featured plots of selected marker gene overlays in Supplemental Figure 5A). Within the macrophages, 8 different clusters were detected. Based on gene markers of existing literature (57), proangiogenesis and proinflammatory macrophages were classified (Supplemental Figure 5, A and B). Within the different macrophage/monocyte clusters, the expression of canonical differentiation markers largely overlapped (Supplemental Figure 5B). All remaining macrophage cluster types failed to pass the classification threshold for any specific macrophage type and stayed as "unassigned." Because CD8 + T cells were unequivocally CXCL10 production. To more specifically ascertain whether the observed effects were a result of deletion of miR-21 in macrophages, we used LysMCre;miR-21 fl/fl mice. LysMCre mice allow for both fairly specific and highly efficient (83%-98%) Cre-mediated deletion of mature macrophages (52).
Deletion of miR-21 in the macrophages also led to the development of smaller tumors when compared with control mice (miR-21 fl/fl ) (Figure 9, A and B). LLC tumors from LysMCre;miR-21 fl/fl mice did not have decreased TAM infiltration; however, their MHCII expression was increased ( Figure 9C). Tumors from LysMCre; miR-21 fl/fl mice showed increased levels of cell death ( Figure 9D), and although the overall number of CD8 + T cells was not affected, they had higher levels of GZMB and extracellular CD107a ( Figure  9E). Tumors from LysMCre;miR-21 fl/fl mice also exhibited reduced neovascularization ( Figure 9F), which was associated with reduced proliferating TECs ( Figure 9G) and an increased percentage of TUNEL + CD31 + TECs(supplemental Figure 4A). This effect could also be attributable to the higher levels of CXCL10 found in CD68 + macrophages of these tumors ( Figure 9H).
To ascertain whether these effects on tumor growth were due to LLCs being grown in a non-organ-specific microenvironment, we performed orthotopic implantation of the isogenic LL/2 Red-Fluc murine LLC-derived cell line (Figure 10, A and B) or isogenic B16 melanoma murine skin cancer cell line (Supplemental Figure  4, B and C) into LysMCre;miR-21 fl/fl and analyzed tumor growth. Deletion of miR-21 in macrophages led to the development of smaller tumors in these 2 different orthopic models. When cancer cells were implanted in an organ-specific microenvironment, the effect of miR-21 deficiency in macrophages was exacerbated (Figure 9, A and B, and Figure 10B). Some reports have described LysM-mediated Cre recombination in DCs, although efficiencies range from 5% to 50% depend-  Figure  11D). Prediction network analysis of these regulated genes showed IL-12 as a potential upstream regulator, while degranulation and cytotoxicity of T cells were predicted as downstream functions of these gene expression changes ( Figure 11E). Importantly, block-identified, we analyzed the changes in gene expression in CD8 + TILs (with intact miR-21) that were either in an immune infiltrate environment mostly made up of macrophages expressing miR-21 (miR-21 fl/fl ) or not expressing miR-21 (LysMCre;miR-21 fl/fl ). Figure  11C shows a dot plot analysis of differentially expressed genes in CD8 + T cells of the tumor immune infiltrate of miR-21 fl/fl or LysMCre; miR-21 fl/fl mice. Interestingly, the proportion of cells with high expression of genes associated with an activated and cytotoxic phenotype, including Il12rb2, Cd69, Ifng, Ccl4, Ccl3, Cxcl10, and and C). Interestingly, splenic macrophages did not show any pHLIP Var3-AF546 cell incorporation. When compared with other cells of the tumor immune infiltrate, also in an acidic microenvironment, TAMs seem to be more efficient at incorporating pHLIP Var3-AF546 because a greater number of cells showed a higher amount of inserted peptide when compared with CD4 + or CD8 + T cells and DCs (Supplemental Figure 6F). Interestingly, nonimmune cells of the TME, including ECs did not efficiently internalize pHLIP when compared with CD68 + macrophages within the tumor (Supplemental Figure 6F). These data indicate that pHLIP is an efficient way to target not only cancer cells but also TAMs. We then assessed the therapeutic antitumor efficacy of pHLIP anti-miR-21 PNA in vivo. The PNAs were also tagged with TAMRA for visualization. Intravenous administration of pHLIP anti-miR-21 to mice with orthotopic and heterotopic lung cancer tumors resulted in a significant reduction in tumor growth when compared with mice injected with pHLIP anti-miR-mismatch control (Supplemental Figure 8, A and B, and Figure 13A). Furthermore, enhanced delivery (TAMRA fluorescence) to macrophages within the tumor was detected by flow cytometry and by immunofluorescence in CD68 + macrophages (Supplemental Figure 6, D and E, respectively). Despite the fact that DCs also became glycolytic upon activation (64), we only found a small proportion of DCs that showed low levels of incorporation of pHLIP Var3-AF546 (Supplemental Figure  6, A-C) or pHLIP anti-miR-21-TAMRA (Supplemental Figure 6, D and E). This was not sufficient to reduce miR-21 levels in DCs or affect miR-21 target expression (Supplemental Figure 6G).
To avoid targeting cancer cells expressing miR-21, we used CRISPR/Cas9-mediated genome editing to KO miR-21 from LLC cells (miR-21 -/-LLCs) (Supplemental Figure 7). The effect of pHLIP anti-miR-21 was assessed in mice bearing miR-21 -/-LLC tumors ( Figure 13B). pHLIP anti-miR-21 administration still reduced tumor burden in mice bearing miR-21 -/-LLC tumors, suggesting that targeting miR-21 in TAMs is sufficient to efficiently diminish tumor growth. Moreover, increased tumor cell death and reduced neovascularization were also observed ( Figure 13, C and D). mRNA levels of miR-21 targets such as Il12, Tnf, or Cxcl10 were increased in sorted TAMs from miR-21 -/-LLC tumors that were administered pHLIP anti-miR-21 ( Figure 13E), indicating efficient miR-21 inhibition in TAMs. We also observed improved T-cell responses as indicated by increased GZMB and degranula-ade of IL-12 and CXCL10 in tumor-bearing LysMCre;miR-21 fl/fl mice produced an increase in tumor growth as compared with LysMCre;miR-21 fl/fl mice treated with an isotype control ( Figure 12, A and B). Furthermore, we observed a reduction in the expression of GZMB in CD8 + TILs when IL-12 and CXCL10 were neutralized in LysMCre;miR-21 fl/fl mice ( Figure 12C), as well as an increase in neovascularization ( Figure 12D). On the whole, our results provide evidence that the decreased tumor growth, improved CTL response, and decreased neovascularization observed in animals lacking miR-21 in TAMs is mediated, at least in part, by increased IL-12 and CXCL10.
Antagonism of miR-21 in macrophages reduces tumor progression even when miR-21 is not expressed in cancer cells. Given the desirable outcomes that miR-21 deficiency in macrophages produces, we aimed to specifically inhibit miR-21 in macrophages during tumor progression. We took advantage of a pHLIP, which is a carrier peptide that under acidic conditions can insert directionally across cell membranes and thereby translocate otherwise membrane-impermeable cargo molecules into cells via a nonendocytic route (58). Given the acidic conditions found in solid tumors, pHLIP has been shown to home to a variety of tumors when administered systemically (59). pHLIP has been coupled to antisense nucleic acid analogues consisting of peptide nucleic acids (PNAs), to create delivery vectors to successfully silence tumor miRNAs (60). The acidic environment found in the tumor has been largely attributed to the high glycolytic rate of tumor cells (61). TAMs have also activated aerobic glycolysis and thus contribute to a local acidification within the TME (62). We wondered whether pHLIP would also efficiently accumulate in TAMs in addition to tumor cells. Enhanced insertion of pHLIP into a variety of cells has been shown under acidic conditions in vitro (58). However, internalization of pHLIP by different cell types within the tumor has not been reported. Thus, we s.c. implanted eGFP-LLCs, and 2 weeks, later mice were injected with pHLIP Variant 3 (Var3) conjugated with fluorescent Alexa546 (63). Single cells of the tumor were isolated, and Alexa546 fluorescence was analyzed via flow cytometry. We found that in addition to cancer cells (eGFP LLCs), the fluorescent signal from pHLIP Var3-AF546 was also detected in TAMs (Supplemental Figure 6, A-C). Although the percentage of cancer cells that incorporated pHLIP Var3-AF546 was higher (Supplemental Figure 6A), the cell-associated fluorescence was greater in TAMs (Supplemental Figure 6, B I NSLC show that high expression of miR-21 in the nontumor cells of the TME is significantly associated with reduced overall survival. In agreement with these findings, a recent report shows that increased expression of miR-21 in the TME, but not in cancer cells, was associated with a poor prognosis for patients with lung adenocarcinoma (31). This effect was linked with increased expression of miR-21 in cancer-associated fibroblasts (CAFs) (31), similar to what has been reported in pancreatic and colorectal tumors (21)(22)(23). However, the underlying mechanisms of cancer-associated fibroblast-miR-21 promoting tumor growth were only investigated in vitro. We show that the expression of miR-21 in infiltrated immune cells, not stromal cells, is responsible for promoting tumor growth. This finding was supported by the decreased tumor growth in WT mice adoptively transferred with miR-21 -/-BM, whereas reverse BMT (WT BM to miR-21 -/recipients) produced a converse effect. This observation was in agreement with a recent study that also used BM adoptive transplant to show that deletion of miR-21 in immune cells reduces tumor growth (25). Elevation of miR-21 levels has been observed in MDSCs directly derived from BM cells that were stimulated with GM-CSF and IL-6, as well as MDSCs from tumor-bearing mice (19), and in vitro overexpression or inhibition of miR-21 in BM cells altered MDSC expansion (19). However, within the tumor infiltrate of miR-21 -/or WT mice adoptively transferred with miR-21 -/-BM, we did not find differences in the proportion of monocytic or granulocytic MDSCs when compared with controls. This finding indicates that the in vivo absence of miR-21 in MDSCs does not have a major effect on this population. Similarly, when we analyzed TILs, we did not find differences in the percentage of IFNG expressing CD4 + or CD8 + tion in CD8 + TILs ( Figure 13F). Furthermore, increased levels of CXCL10 in CD68 + macrophages of these tumors was associated with reduced CD31 + capillary structures ( Figure 13G).
Taken together, these results show that targeting miR-21 in macrophages promotes an antitumoral immune response and an overall proinflammatory angiostatic function that results in a significant reduction in tumor growth.

Discussion
In this study, we have used a variety of different mouse models to assess how miR-21 in noncancerous cells of the TME contributes to tumor progression. Cancer initiation and progression has been historically defined by the behavior of cancer cells, including regulation by miRNAs, which have an impact on many of the autonomous functions of cancer cells to either promote or suppress tumor growth (1). Importantly, miRNAs in cells from the TME have also emerged as key players involved in the development and progression of cancer (5)(6)(7)65).
Our results using syngeneic heterotopic and orthotopic lung cancer models demonstrate that miR-21 expression in noncancerous cells plays an important role in tumor progression, in agreement with reports using other models (24,25). However, the existing literature fails to fully elucidate the cell compartment of the TME in which miR-21 primarily contributes to tumor progression, often relying on in vitro systems or approaches that globally target miR-21 in a non-cell/tissue-specific manner (19)(20)(21)(22)(23)(24)(25)31).
Within the TME, miR-21 has been reported to be increased in many types of cancer (22,27,31,66), and this increase was linked to tumor progression. Our data, in 3 cohorts of patients with stage LysMCre;miR-21 fl/fl also showed increased levels of cell death and their CD8 + T cells (expressing miR-21). They also had higher levels of GZMB and extracellular CD107a, suggesting that the absence of miR-21 in TAMs can influence other cells within the TME.
Our present results indicate that the observed changes in the levels of chemokines and cytokines in miR-21-deficient TAMs are critical for CTL activation and antitumor activity. Specifically, we found that increased IL-12 in miR-21 -/-TAMs is responsible for the increased GZMB levels independent of miR-21 expression in CD8 + T cells. This finding was supported by our single-cell RNA-Seq analysis, where we analyzed the changes in gene expression in CD8 + TILs from tumors in which TAMs lacked miR-21. In LLC tumors from LysMCre;miR-21 fl/fl mice, the CD8 + TILs exhibit increased expression of genes associated with an activated and cytotoxic phenotype, including Il12rb2, Cd69, Ifng, Ccl4, Ccl3, Cxcl10, and several granzyme coding genes. These results provide evidence that the improved CTL response is the result of miR-21 deletion in macrophages.
In addition to the effects on T-cell functions, the increased levels of IL-12 in LysMCre;miR-21 fl/fl mice could also play an important role in regulation of tumor angiogenesis, as IL-12 has been shown to negatively regulate tumor neovascularization (50). This effect has been associated with IFN-inducible production of CXCL10 and CXCL9 (51). Macrophages lacking miR-21 exhibited an increased production of CXCL10, which binds to the CXCR3-B receptor on endothelial cells, resulting in suppression of endothelial cell proliferation and differentiation into capillary structures (51). CXCL10 selectively attracts Th1 cells and functions as a positive feedback loop in the Th1-driven antitumor immune response (45). The tumors from LysMCre;miR-21 fl/fl mice exhibited reduced neovascularization, a reduced number of proliferating TECs, and an increased percentage of TUNEL + CD31 + TECs. Taken together, our present work indicates that the absence of miR-21 in macrophages favors the formation of a tumor-suppressive microenvironment (45), characterized by increased expression of IL-12 and CXCL10 that leads to improved CTL response, decreased neovascularization, and decreased tumor burden.
Macrophages are perhaps the most important immune cells that infiltrate the tumor and affect its pathology (71,72). Although macrophages can show some antitumor activity (73), they mostly promote a net protumor outcome because they have been shown to be capable of dampening T-cell responses and stimulating angiogenesis, among others actions (47,74). This phenomenon, which has been widely accepted, is promoted by tumor cells that create a widespread tolerogenic environment through the release of tumor-derived factors that promote the expansion and reprogramming of TAMs to block T-cell functions and promote several protumor events. TAMs are recognized as an important target for therapeutic intervention (75). Our data highlight the central role of miR-21 in regulation of TAM activity and function regarding CTL regulation and neovascularization. Thus, to determine whether the high expression of miR-21 in TAMs may make it a likely target for therapeutic intervention, we opted to use pHLIP anti-miR-21 PNA. pHLIP carriers coupled to antisense nucleic acid analogues are delivery vectors that have been shown to successfully target miRNAs in tumor cells (60,76). Here, we show for the first time that TAMs incorporate pHLIP as efficiently as tumors cell and T cells, in agreement with a recent report (25). In other settings, miR-21 has been shown to regulate cell-autonomous T-cell function (67,68). In the context of tumor progression, a report showed that absence of miR-21 reduced the proliferation of both CD4 + and CD8 + cells and their cytokine production, thus accelerating the growth of grafted tumors (24). In this case, growth of an implanted S180 mouse cancer cell line was analyzed in miR-21 floxed expressing the global Cre driver. S180 cells have been shown to grow in multiple inbred mouse strains due to β2-microglobulin deficiency, MHC class I destabilization, and impaired recognition by host CTLs (69). In this setting and without using a syngeneic mouse model and/or conditional deletion of miR-21 in T cells, it was concluded that miR-21 expression in T cells was protective against tumor growth (24).
Several studies have described correlations between macrophage number, location, and phenotypic characteristics and clinical outcomes (70). In our human TMAs, we found that high expression of miR-21 in CD68 + macrophages within the TME compartment had a negative impact in patient disease-specific survival and shows the relevance of miR-21 in macrophages for tumor progression. In line with miR-21 being the most abundant miRNA in macrophages (10), we found that within the LLC tumors, miR-21 is highly expressed in TAMs compared with the rest of the tumor immune infiltrate. Because conflicting results have been reported regarding the role of miR-21 in regulating the inflammatory responses of macrophages (10, 13-15, 17, 18, 43), we specifically focused on how miR-21 regulates the gene expression and functions of TAMs. miR-21 deficiency in TAMs produced a global rewiring of their transcriptional regulatory network that is skewed toward a proinflammatory phenotype and the promotion of an antitumoral immune response. Importantly, we show that selective deletion of miR-21 in macrophages in a heterotopic and orthotopic model of lung cancer mimicked the effects of global or hematopoietic loss of miR-21, with smaller tumors, increased cell death, and reduced neovascularization. Moreover, we found increased GZMB and degranulation of CD8 + TILs, indicating increased CTL activity and heightened killing capacity, consistent with the higher levels of cell death within the tumors of LysMCre;miR-21 fl/fl mice. These findings support the report of Baer and colleagues who showed that disruption of miRNA processing in TAMs, by LysM-Cre-mediated deletion of Dicer1, leads to the development of smaller LLC tumors and improved CTL activity (5). This study highlights how the loss of mature miRNAs in TAMs during tumor progression leads to increased classically activated TAMs, improved CD8 response, and decreased CD8 suppression, which was associated with higher expression of CXCL10, CD86, and CXCL9 in the tumors (5). In fact, because miR-21 is highly expressed in macrophages (10), many of these changes could be attributed to the loss of expression of miR-21 after Dicer1 deletion.
A recent report using a B16 tumor model suggested that mice receiving miR-21-deficient BM had higher numbers of "M1" TAMs with tumoricidal polarization, whereas miR-21 deficiency in T cells reduced their ability to produce proinflammatory cytokines and cytolytic granules (25). On the contrary, our data indicate that CD8 + TILs from miR-21 -/mice have increased GZMB expression and killing capacity. However, the increased GZMB expression is not due to miR-21 deletion in these cells because LLC tumors from jci.org Volume 129 Number 12 December 2019 roni correction for multiple comparisons. Normality was checked using the Kolmogorov-Smirnov test. A nonparametric test, Mann-Whitney U, test or a Kruskal-Wallis test for multiple comparisons, was used when data did not pass the normality test. For RNA-Seq analysis, we used Benjamini-Hochberg False Discovery Rate. Survival curves were generated by Kaplan-Meier analysis and tested for significance using the Mantel-Cox log-rank test. A value of P ≤ 0.05 was considered statistically significant. Data analysis was performed using GraphPad Prism Software Version 7. Study approval. Animal experiments were conducted under the ethical guidelines and protocols approved by IACUC at Yale University School of Medicine (Animal protocol no. 2019-116576). All human tissues were collected with the approval from the Yale Human Investigation Committee (protocol no. 9505008219). The Yale Human Investigation Committee approved the patient consent forms or in some cases, a waiver of consent.

Author contributions
MS, BC, and JS assisted with experimental design, performed experiments, analyzed data, and interpreted results. YL, RGM, and SM analyzed data. WD performed experiments. YKR provided pHLIP (Var3). AK, SO, and RB synthesized miR-21 PNA and conjugated to pHLIP. NLP analyzed data and edited the manuscript. PF provided LysMCre;miR-21 fl/fl mice. PMG, DLR, and CF-H assisted with experimental design and data interpretation. YS designed the study, analyzed data, interpreted results, and wrote the manuscript (comments were added by all authors). strikingly more effectively than other cells of the TME. pHLIP anti-miR-21 administration in WT mice reduced tumor burden even under conditions where tumor cells lacked miR-21 expression. Importantly, CD8 + TILs showed increased GZMB expression and tumors had reduced neovascularization. Sorted TAMs of pHLIP anti-miR-21 treated mice exhibited increased IL-12 and CXCL10 expression, similar to TAMs of LysMCre;miR-21 fl/fl mice. Thus, targeted inhibition of miR-21 in TAMs is sufficient to increase CTL activity and decrease angiogenesis, thereby diminishing tumor growth.
Numerous studies have shown that within cancer cells, miR-21 acts as an oncomiR, and anti-miR-21 therapy (RG-012) is already in the pipeline for clinical trials. Our findings demonstrate the critical role of miR-21 within TAMs for promoting tumor development, indicating that targeting of miR-21 may have the added benefit of improving CTL activity and limiting tumor angiogenesis through its effects on TAMs. Our findings in human patients with NSCLC suggest this may have a critical impact on patient outcomes. Finally, our results indicate that use of pHLIP targeted therapies allows for effective targeting of both tumor cells and TAMs, while avoiding possible unintended effects on other tissues and organs.