Anti-tumor Macrophages Research Articles

Abstract 206: Intratumoral accumulation of CD38 enhances immune evasion and metastasis in models of breast cancer with EMT plasticity

Abstract Background- Triple-negative breast cancer (TNBC) is challenging to treat due to fewer clinically detectable targets and its propensity to metastasize. Epithelial to mesenchymal transition (EMT) is a key feature of the metastatic cascade. Recent data highlights the enhanced metastatic potential of hybrid epithelial-mesenchymal (EM) phenotypes from partial activation of EMT. However, mechanistic insights and targetable vulnerabilities within hybrid EM remain elusive. We identified that hybrid EM tumors are enriched in CD38, an immunesuppressive molecule associated with worse clinical outcomes in liquid malignancies. Hence, we sought to investigate role of CD38 in hybrid EM-driven metastasis. We hypothesize that hybrid EM tumors drive metastasis via intratumoral accumulation of CD38, promoting an immunosuppressive tumor microenvironment (TME). Methods- Reverse Phase Protein Array, cell migration/invasion assays, flowcytometry and immunohistochemistry were used to evaluate phenotype, immune cell populations and molecular mechanisms upon CD38 knockdown (KD) and overexpression (OE) in vivo. Results- CD38 KD weakened the process of EMT and reduced cell migration and invasion. Tumors lacking CD38 showed increased infiltration of CD8 T cells and M1-like (anti-tumor) macrophages coupled with a decrease in immunosuppressive regulatory T cells (Tregs) and M2-like (pro-tumor) macrophages. In contrast, cancer cells with CD38 OE maintained a hybrid phenotype through co-expression of epithelial and mesenchymal markers and also showed significant increase in cell migration and invasion. Tumors arising from CD38 OE cells displayed a significant increase in Tregs. Most strikingly, hybrid EM tumors that no longer express CD38 show reduction in lung metastases and circulating tumor cell colonies. Taken together, these findings suggest that CD38 is important for the metastatic and immune evasive potential of hybrid EM breast cancers. Conclusion- Our data exposes CD38 as a survival strategy in hybrid EM tumors to suppress immune cells and sustain metastasis with strong implications in other carcinomas which have hybrid EM properties. Citation Format: Tanvi Visal. Intratumoral accumulation of CD38 enhances immune evasion and metastasis in models of breast cancer with EMT plasticity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 206.

ICOS costimulation in combination with CTLA-4 blockade remodels tumor-associated macrophages toward an antitumor phenotype.

We have previously demonstrated synergy between ICOS costimulation (IVAX; ICOSL-transduced B16-F10 cellular vaccine) and CTLA-4 blockade in antitumor therapy. In this study, we employed CyTOF and single-cell RNA sequencing and observed significant remodeling of the lymphoid and myeloid compartments in combination therapy. Compared with anti-CTLA-4 monotherapy, the combination therapy enriched Th1 CD4 T cells, effector CD8 T cells, and M1-like antitumor proinflammatory macrophages. These macrophages were critical to the therapeutic efficacy of anti-CTLA-4 combined with IVAX or anti-PD-1. Macrophage depletion with clodronate reduced the tumor-infiltrating effector CD4 and CD8 T cells, impairing their antitumor functions. Furthermore, the recruitment and polarization of M1-like macrophages required IFN-γ. Therefore, in this study, we show that there is a positive feedback loop between intratumoral effector T cells and tumor-associated macrophages (TAMs), in which the IFN-γ produced by the T cells polarizes the TAMs into M1-like phenotype, and the TAMs, in turn, reshape the tumor microenvironment to facilitate T cell infiltration, immune function, and tumor rejection.

Abstract 5365: Characterizing signaling in macrophage-GBM cell coculture

Abstract Macrophages are a type of immune cell that play a crucial role in the tumor microenvironment. In tumors, macrophages can be polarized towards a pro-inflammatory phenotype, referred to as M1, or towards an anti-inflammatory and tumor-promoting phenotype, referred to as M2. M2 macrophages promote tumor growth by secreting cytokines that increase tumor proliferation, angiogenesis, and dampen the immune response from other immune cells such as T cells and dendritic cells. The balance between M1 and M2 macrophages in the tumor microenvironment is critical in determining the outcome of cancer progression and response to therapy. By understanding the pathways that lead to macrophage polarization, we can develop therapeutics that inhibit this process and support an anti-tumor macrophage response. Co-culture is a powerful model to study interactions between different cell types. We created co-cultures with macrophages and glioblastoma multiforme (GBM) cells to understand how these cell-cell interactions can lead to altered cell phenotype. Several studies have looked at the interaction of two populations using a transwell system where one cell population is placed in a well with a permeable membrane and the other population is placed below, allowing for the exchange of soluble factors. However, this does not account for an important mode of cell-cell interaction, which is physical contact. Therefore, we tested how macrophages derived from primary monocytes polarized in the context of conditioned media (supernatant), cell lysate, and direct co-culture. We used U87-MG as a model GBM cell line. We assayed the M2 phenotype by using flow cytometry with CD163 as a marker. Our findings show that direct co-culture is crucial for promoting macrophage M2 polarization. Using our in vitro model for tumor-induced macrophage polarization, we conducted phosphoproteomic analysis to characterize signals associated with this phenotypic change. To study how co-culture induced differential signaling in tumor populations and macrophages, we devised a method to fix cells to preserve cell signaling, followed by separation using flow cytometry and LC-MS/MS analysis of individual cell populations. We created co-cultures of U87-MG tumor cells and macrophage-differentiated THP-1 cells, a monocyte cell line. We then tested whether our method could reliably separate the different populations, whether we could detect altered signals due to co-culture, and showcase how separating cell populations yields information that could not be gathered from bulk analysis of co-cultures. Our data shows that the analysis was highly reproducible and that the signaling from cells separated from co-culture was highly similar to signaling in cells that were grown individually. However, some phosphorylation residues were uniquely upregulated in the co-culture condition, pointing to signaling that may occur in response to tumor interaction. Citation Format: Alicia D. D'Souza, Rachit Mukkamala, Ryuhjin Ahn, Forest M. White. Characterizing signaling in macrophage-GBM cell coculture [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5365.

Abstract 6532: Blockade of CD24 sensitizes pancreatic cancer to Kras inhibitors

Abstract Pancreatic ductal adenocarcinoma (PDAC) is predominantly driven by KRAS oncogene. KRAS, long considered an undruggable target, has been targeted by drugs including a KRASG12C inhibitor (G12Ci), AMG 510, which is approved for the treatment of KRASG12C-mutant non-small cell lung cancer (NSCLC). AMG 510 also exhibited promising anticancer activity in human PDAC, with 21% response rate and over 70% patients experiencing certain level of tumor control. However, targeting KRAS oncogenes faces the major challenge of therapy resistance, which severely limited its survival benefit. While multiple studies have elucidated potential resistance mechanisms to G12Ci in lung cancer, the molecular mechanisms underlying resistance in PDAC remains elusive. Here, using KRASG12C-driven mouse PDAC model, we found that similar to the clinical observation, KRASG12C tumors exhibit only partial response to AMG 510 treatment. CyTOF analysis indicates that such tumor persistence following G12Ci treatment occurs even in the presence of conversion from a ‘cold’ pro-tumor immune microenvironment to a ‘hot’ anti-tumor immune environment, as evidenced by the prominent increase in both cytotoxic T cells and M1-like macrophages. Interestingly, we discovered that the expression of CD24, which was recently identified as a new ‘do not eat me’ signal, is significantly upregulated on the surface of persist tumor cells after G12Ci treatment which suppresses the function of anti-tumor macrophages. Anti-CD24 blocking mAb enhanced macrophage-mediated phagocytosis in vitro and significantly sensitizes KRASG12C tumors to AMG 510 treatment in vivo in a macrophage-dependent manner. Similar findings were observed in KRASG12D-driven PDAC model. Our findings identified a novel immune combination to improve the outcome of KRAS targeted therapy in PDAC. Citation Format: Yongkun Wei, Minghui Liu, Er-Yen Yen, Jun Yao, Phuoc T. Nguyen, Xiaofei Wang, Zecheng Yang, Abdelrahman Yousef, Dean Pan, Yanqing Jin, Madelaine S. Theady, Jangho Park, Yiming Cai, Mitsunobu Takeda, Matthew Vasquez, Yong Zhou, Hong Zhao, Andrea Viale, Huamin Wang, Dan Zhao, Ronald A. DePinho, Wantong Yao, Haoqiang Ying. Blockade of CD24 sensitizes pancreatic cancer to Kras inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6532.

New Strategies for Macrophage Re-Education in Cancer: An Update.

The association between cancer and inflammation is well established. Chronic inflammation represents a fundamental step in the development and progression of some types of cancer. Tumors are composed of a heterogeneous population of infiltrating cells including macrophages, fibroblasts, lymphocytes, granulocytes, and mast cells, which respond to signals from the microenvironment and, in turn, produce cytokines, chemokines, transcription factors, receptors, and miRNAs. Recent data demonstrate that, in addition to classical (M1) and alternative (M2) macrophage subtypes, there are many intermediate subtypes that potentially play different roles in response to environmental stimuli. Tumors are infiltrated by macrophages called TAMs that mainly display an M2-like phenotype and tumor growth-permissive activities. There is a bidirectional interaction between tumor cells and tumor-infiltrating cells that determines macrophage polarization and ultimately tumor progression or regression. These complex interactions are still unclear but understanding them is fundamental for the development of new therapeutic strategies. Re-educating tumor-permissive macrophages into anti-tumor macrophages is a new focus of research. This review aims to analyze the most recent articles investigating the interplay between tumors, tumor-infiltrating cells, and TAMs, and the strategies for re-educating tumor-permissive macrophages.

Discovery and Preclinical Activity of BMS-986351, an Antibody to SIRPα That Enhances Macrophage-mediated Tumor Phagocytosis When Combined with Opsonizing Antibodies.

Increasing the phagocytotic capabilities of tumor-associated macrophages by modulating macrophage-tumor cell surface signaling via the CD47-SIRPα axis is a novel strategy. Molecules targeting CD47 have potential but its ubiquitous expression necessitates higher therapeutic doses to overcome potential antigen sink effects. The restricted expression pattern of SIRPα may limit toxicities and lower doses of the SIRPα antibody BMS-986351 may overcome target mediated drug disposition while maintaining the desired pharmacology.

Abstract C097: KRASG12C exhibits allele-specific biology in pancreatic cancer and targeting CD24 sensitizes KRASG12C-driven tumors to sotorasib treatment

Abstract Pancreatic adenocarcinoma (PDAC) is predominantly driven by KRAS oncogene. While all oncogenic KRAS mutations result in its hyperactivation, the allele-specific biology of KRAS mutations remain poorly understood. Addressing such issues has become particularly important with the recent advancements in the development of allele-specific and pan-KRAS inhibitors. Among the KRAS inhibitors, KRASG12C inhibitors (G12Ci), Sotorasib and Adagrasib, have been approved for treating KRASG12C-mutant lung cancer. Sotorasib also exhibited promising anticancer activity in human PDAC, with 21% response rate and over 70% patients experiencing certain level of tumor control. However, targeting KRAS oncogenes faces the major challenge of therapy resistance, which severely limited its survival benefit. While multiple studies have elucidated potential resistance mechanisms to G12Ci in lung cancer, the molecular mechanisms underlying its resistance in PDAC remains elusive, largely owing to the scarcity of relevant and faithful preclinical models. Here, we report the generation of a new autochthonous KRASG12C-driven PDAC model (p48Cre; LSL-KrasG12C; p53L/+, KCPC). Compared to the KRASG12D-driven KPC model (p48Cre; LSL-KrasG12D; p53L/+), tumor development in KCPC model is significantly delayed accompanied with corresponding decrease in both KRAS signaling and proliferation in premalignant lesions. As a result, the overall survival of the KCPC model is significantly prolonged compared to the KPC model, indicating allele-specific discrepancy of KRAS mutations during PDAC initiation. However, upon malignant progression, KRASG12C-driven mouse PDAC exhibit similar histopathological and molecular features as KRASG12D-driven tumors. Furthermore, similar to the clinical observation, KCPC tumors exhibit only partial response to Sotorasib treatment. CyTOF analysis indicates that such tumor persistence following G12Ci treatment occurs even in the presence of conversion from a ‘cold’ pro-tumor immune microenvironment to a ‘hot’ anti-tumor immune environment, as evidenced by the prominent increase in both cytotoxic T cells and M1-like macrophages. Interestingly, we discovered that the expression of CD24, which was recently identified as a new ‘do not eat me’ signal, is significantly upregulated on the surface of persist tumor cells, which suppresses the function of anti-tumor macrophages. Treatment with anti-CD24 blocking mAb enhanced macrophage-mediated phagocytosis in vitro and significantly sensitizes KCPC-derived xenograft tumors to Sotorasib treatment in vivo in a macrophage-dependent manner. Similar upregulation of CD24 following KRAS inhibition and therapeutic benefit of co-targeting CD24 and KRAS were also observed in KRASG12D-driven mouse and human PDAC, implicating the potential of such combination in a broader spectrum of KRAS-driven PDAC. Together, our findings revealed the allele-specific function of KRASG12C in the development of PDAC and identified a novel immune combination to improve the outcome of KRAS targeted therapy in PDAC. Citation Format: Yongkun Wei, Minghui Liu, Phuoc T. Nguyen, Dean N. Pan, Wantong Yao, Haoqiang Ying. KRASG12C exhibits allele-specific biology in pancreatic cancer and targeting CD24 sensitizes KRASG12C-driven tumors to sotorasib treatment [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr C097.

Blockade of C5a receptor unleashes tumor-associated macrophage antitumor response and enhances CXCL9-dependent CD8+ T cell activity

Macrophages play a crucial role in shaping the immune state within the tumor microenvironment (TME) and are often influenced by tumors to hinder antitumor immunity. However, the underlying mechanisms are still elusive. Here, we observed abnormal expression of complement 5a receptor (C5aR) in human ovarian cancer (OC), and identified high levels of C5aR expression on tumor-associated macrophages (TAMs), which led to the polarization of TAMs toward an immunosuppressive phenotype. C5aR knockout or inhibitor treatment restored TAM antitumor response and attenuated tumor progression. Mechanistically, C5aR deficiency reprogrammed macrophages from a protumor state to an antitumor state, associating with the upregulation of immune response and stimulation pathways, which in turn resulted in the enhanced antitumor response of cytotoxic Tcells in a manner dependent on chemokine (C-X-C motif) ligand 9 (CXCL9). The pharmacological inhibitionof C5aR also improved the efficacy of immune checkpoint blockade therapy. In patients, C5aR expression associated with CXCL9 production and infiltration of CD8+ Tcells, and a high C5aR level predicted poor clinical outcomes and worse benefits from anti-PD-1 therapy. Thus, our study sheds light on the mechanisms underlying the modulation of TAM antitumor immune response by the C5a-C5aR axis andhighlights the potential of targeting C5aR for clinical applications.

Abstract PR02: Lyn kinase regulates myeloid-cell responses to mammary tumors

Abstract Myeloid-cell enrichment in tumors is correlated with increased metastasis, reduced survival times, and poor treatment outcomes in multiple cancers, including breast cancer. Many signals (CSF, Il-6, chemokine) to myeloid cells in the tumor microenvironment are transduced through the Src-family kinase Lyn. Expression of both isoforms of Lyn (LynA and LynB) is regulated by cytokine signaling and cell type, and total Lyn expression is negatively correlated with breast cancer patient survival. Lyn expression in macrophages contributes to leukemogenesis, but the functional contributions of LynA and LynB in solid tumors have not been studied. We are examining the contributions of Lyn in trafficking of myeloid cells to mammary tumors, pro-tumor polarization of tumor-associated macrophages, and growth and metastasis of mammary tumors. In an E0771 orthotopic transplant model in hosts expressing or lacking LynA or LynB expression, we have discovered that Lyn expression in the mammary tumor microenvironment promotes the early recruitment of monocytes, macrophages, neutrophils, and dendritic cells, as determined by flow cytometry analysis of tumors early in development. Furthermore, Lyn promotes pro-tumor polarization of tumor-associated macrophages. Tumors from Lyn knockout animals contained populations of inflammatory, anti-tumor macrophages that were not significantly different in size from WT, but the CD206+, anti-inflammatory population was reduced. Finally, we have observed that expression of either Lyn isoform in the tumor microenvironment can promote tumor growth and decrease survival in orthotopically transplanted mice. In contrast, our studies suggest that only LynB promotes metastasis to the lungs of transplanted mice, as determined by in vivo imaging of a hyper-metastatic, luciferase-expressing variant of E0771. Together, our findings suggest that Lyn kinase plays a role in the signaling response of myeloid cells, which are major factors in disease course and treatment response, to tumor-associated stimuli. Modulation of Lyn activity, specifically the LynB isoform, or the activity of its downstream signaling partners, is thus a potential strategy for influencing the trafficking and polarization of myeloid cells within breast tumors to benefit patients. Citation Format: Joseph T Greene, Ben F Brian, Aditi Bapat, Tanya S Freedman. Lyn kinase regulates myeloid-cell responses to mammary tumors [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr PR02.

Ultrasound-responsive spherical nucleic acid against c-Myc/PD-L1 to enhance anti-tumoral macrophages in triple-negative breast cancer progression.

Triple-negative breast cancer (TNBC) is the most challenging breast cancer subtype because of its aggressive behavior and limited therapeutic targets. c-Myc is hyperactivated in the majority of TNBC tissues, however, it has been considered an "undruggable" target due to its disordered structure. Herein, we developed an ultrasound-responsive spherical nucleic acid (SNA) against c-Myc and PD-L1 in TNBC. It is a self-assembled and carrier-free system composed of a hydrophilic small-interfering RNA (siRNA) shell and a hydrophobic core made of a peptide nucleic acid (PNA)-based antisense oligonucleotide (ASO) and a sonosensitizer. We accomplished significant enrichment in the tumor by enhanced permeability and retention (EPR) effect, the controllable release of effective elements by ultrasound activation, and the combination of targeted therapy, immunotherapy and physiotherapy. Our study demonstrated significant anti-tumoral effects in vitro and in vivo. Mass cytometry showed an invigorated tumor microenvironment (TME) characterized by a significant alteration in the composition of tumor-associated macrophages (TAM) and decreased proportion of PD-1-positive (PD-1+) T effector cells after appropriate treatment of the ultrasound-responsive SNA (USNA). Further experiments verified that tumor-conditioned macrophages residing in the TME were transformed into the anti-tumoral population. Our finding offers a novel therapeutic strategy against the "undruggable" c-Myc, develops a new targeted therapy for c-Myc/PD-L1 and provides a treatment option for the TNBC.

Fe-doped carbon dots: a novel biocompatible nanoplatform for multi-level cancer therapy

BackgroundTumor treatment still remains a clinical challenge, requiring the development of biocompatible and efficient anti-tumor nanodrugs. Carbon dots (CDs) has become promising nanomedicines for cancer therapy due to its low cytotoxicity and easy customization.ResultsHerein, we introduced a novel type of “green” nanodrug for multi-level cancer therapy utilizing Fe-doped carbon dots (Fe-CDs) derived from iron nutrient supplement. With no requirement for target moieties or external stimuli, the sole intravenous administration of Fe-CDs demonstrated unexpected anti-tumor activity, completely suppressing tumor growth in mice. Continuous administration of Fe-CDs for several weeks showed no toxic effects in vivo, highlighting its exceptional biocompatibility. The as-synthesized Fe-CDs could selectively induce tumor cells apoptosis by BAX/Caspase 9/Caspase 3/PARP signal pathways and activate antitumoral macrophages by inhibiting the IL-10/Arg-1 axis, contributing to its significant tumor immunotherapy effect. Additionally, the epithelial-mesenchymal transition (EMT) process was inhibited under the treatment of Fe-CDs by MAPK/Snail pathways, indicating the capacity of Fe-CDs to inhibit tumor recurrence and metastasis.ConclusionsA three-level tumor treatment strategy from direct killing to activating immunity to inhibiting metastasis was achieved based on “green” Fe-CDs. Our findings reveal the broad clinical potential of Fe-CDs as a novel candidate for anti-tumor nanodrugs and nanoplatform.

Nanomedicine-based co-delivery of a calcium channel inhibitor and a small molecule targeting CD47 for lung cancer immunotherapy

Pro-tumoral macrophages in lung tumors present a significant challenge in immunotherapy. Here, we introduce a pH-responsive nanomedicine approach for activating anti-tumoral macrophages and dendritic cells. Using a layered double hydroxide nanosheet carrier, we co-deliver a T-type calcium channel inhibitor (TTA-Q6) and a CD47 inhibitor (RRX-001) into lung tumors. In the tumor acidic environment, TTA-Q6 is released, disrupting cancer cell calcium uptake, causing endoplasmic reticulum stress and inducing calreticulin transfer to the cell surface. Surface calreticulin activates macrophages and triggers dendritic cell maturation, promoting effective antigen presentation and therefore activating antitumor T cells. Simultaneously, RRX-001 reduces CD47 protein levels, aiding in preventing immune escape by calreticulin-rich cancer cells. In lung tumor models in male mice, this combined approach shows anti-tumor effects and immunity against tumor re-exposure, highlighting its potential for lung cancer immunotherapy.

The Peripheral Blood Stem Cell Immunome Demonstrates Abnormal Immune Effector Cells Early in the Disease Course of Multiple Myeloma and Contributes to Inferior Outcomes and Secondary Myeloid Neoplasms

Background: Upfront autologous stem cell transplant (ASCT) is the standard-of-care for newly diagnosed multiple myeloma (MM) patients. However, relapse and the development of therapy-related myeloid neoplasms (t-MN) post ASCT results in considerable morbidity and mortality. We hypothesized that enrichment of abnormal myeloid progenitors and immune effector cells (IEC) in peripheral blood stem cells (PBSCs) of patients with MM is associated with a higher risk of relapse and development of t-MN. Methods: After institutional review board approval, we screened patients with active MM that were evaluated at Mayo Clinic, Rochester, MN between 01/01/2003 and 12/31/2020. Patients that underwent an ASCT and had cryopreserved PBSC product available for research were included. Using mass cytometry, we performed a comprehensive immunophenotyping of mobilized cryopreserved PBSCs from 54 MM patients undergoing 1 st ASCT, using validated antibody panel of 37 lymphoid- and myeloid-based markers each. Flow cytometry standard files were normalized and concatenated using the Fluidigm acquisition software. Flow cytometry standard files were uploaded to the OMIQ software from Dotmatics (www.omiq.ai, www.dotmatics.com), where transformation and cleaning (doublets, debris) were performed. To correct for between-sample technical variations (“batch effects”), we used fdaNorm within the omiq.ai platform. Clustering and visualization of normalized and concatenaded flowcytometry files were performed within the omiq.ai platform using PhenoGraph and UMAP, respectively. We performed principal component analysis (PCA) to identify major immunomic drivers of variability across patients. We explored the associations between immune subsets and the dichotomized clinical outcomes of interest: progression-free (PFS), myeloid neoplasm-free (MNFS) and overall survival (OS) from ASCT. A 2-sided false discovery rate adjusted P-value of <0.05 was considered significant when multiple comparisons were performed; otherwise, a P-value of <0.05 was considered significant. Results: The most abundant cell populations were T-cells (49% of the CD45 +) and myeloid cells (43% of the CD45 + cells). Clustering on the CD45 + cells revealed 30 unique lymphoid clusters and 27 unique myeloid clusters. Subclustering on NK-cells identified an additional 16 NK-cell clusters. On PCA, principal components-1 (PC-1) and 2 (PC-2) explained 15.2% and 12.5% of the variability in the data, respectively. A high PC-2 score was associated with inferior median PFS (20 vs. 52 months; P=0.005, Figure 1A), MNFS (52 vs. 85 months, P=0.03, Figure 1B) and OS (64 vs. 106 months, P=0.01) from ASCT. Immune subsets enriched in the high PC-2 cohort included the ‘NKT-like’ (CD56 +) T-cell subset which expressed the inhibitory CD159a receptor and TIGIT; a terminally differentiated (CD27/CD28 -, CD57/KLRG1 +) and exhausted (TIGIT/PD-1 +) T cell subset, 2 exhausted (TIGIT/PD-1 +) T cell subsets; an immunosenescent (CD27/CD28 -, CD57/KLRG1 +) T cell subset as well as a terminally exhausted (C27/C28 -, KLRG1/PD-1/TIGIT +) TCRgamma-delta subset. Immune subsets with high loadings within PC-1 (associated with improved clinical outcomes) consisted exclusively of CD4 + cells with early memory phenotypes (CD27/CD28/CD127 +). Abnormal expression of CD7 and HLA-DR expression on myeloid progenitor cell subsets was associated with an inferior PFS, MNFS, and OS. A NK-cell subcluster, characterized by an immature (CD56 high), inhibitory (CD159a +, NKG2A) phenotype, was enriched in patients developing t-MN (0.33% vs. 0.17%, P=0.006). Conclusion: We identify abnormal myeloid and immune effector cells in the PBSCs of patients with MM, even prior to ASCT. Patients with a PBSC product that is enriched in immunosuppressive T and myeloid cell subsets, and with a lower proportion of antigen presenting cells, progenitor populations, and anti-tumor macrophages have a shorter PFS and MNFS. This suggests that the predisposition to t-MN and inferior outcomes for MM are possibly determined early in the disease course. These findings need external validation and have the potential to guide sequencing of future treatment modalities.

Inhibition of murine colorectal cancer metastasis by targeting M2-TAM through STAT3/NF-kB/AKT signaling using macrophage 1-derived extracellular vesicles loaded with oxaliplatin, retinoic acid, and Libidibia ferrea

Colorectal cancer is still unmanageable despite advances in target therapy. However, extracellular vesicles (EVs) have shown potential in nanomedicine as drug delivery systems, especially for modulating the immune cells in the tumor microenvironment (TME). In this study, M1 Macrophage EVs (M1EVs) were used as nanocarriers of oxaliplatin (M1EV1) associated with retinoic acid (M1EV2) and Libidibia ferrea (M1EV3), alone or in combination (M1EV4) to evaluate their antiproliferative and immunomodulatory potential on CT-26 and MC-38 colorectal cancer cell lines and prevent metastasis in mice of allograft and peritoneal colorectal cancer models. Tumors were evaluated by qRT-PCR and immunohistochemistry. The cell death profile and epithelial-mesenchymal transition process (EMT) were analyzed in vitro in colorectal cancer cell lines. Polarization of murine macrophages (RAW264.7 cells) was also carried out. M1EV2 and M1EV3 used alone or particularly M1EV4 downregulated the tumor progression by TME immunomodulation, leading to a decrease in primary tumor size and metastasis in the peritoneum, liver, and lungs. STAT3, NF-kB, and AKT were the major genes downregulated by of M1EV systems. Tumor-associated macrophages (TAMs) shifted from an M2 phenotype (CD163) to an M1 phenotype (CD68) reducing levels of IL-10, TGF-β and CCL22. Furthermore, malignant cells showed overexpression of FADD, APAF-1, caspase-3, and E-cadherin, and decreased expression of MDR1, survivin, vimentin, and PD-L1 after treatment with systems of M1EVs. The study shows that EVs from M1 antitumor macrophages can transport drugs and enhance their immunomodulatory and antitumor activity by modulating pathways associated with cell proliferation, migration, survival, and drug resistance.

In vivo transfection of cytokine genes into tumor cells using a synthetic vehicle promotes antitumor immune responses in a visceral tumor model.

The tumor microenvironment (TME) strongly affects the clinical outcomes of immunotherapy. This study aimed to activate the antitumor immune response by manipulating the TME by transfecting genes encoding relevant cytokines into tumor cells using a synthetic vehicle, which is designed to target tumor cells and promote the expression of transfected genes. Lung tumors were formed by injecting CT26.WT intravenously into BALB/c mice. Upon intravenous injection of the green fluorescent protein-coding plasmid encapsulated in the vehicle, 14.2% tumor-specific expression was observed. Transfection of the granulocyte-macrophage colony-stimulating factor (GM-CSF) and CD40 ligand (L)-plasmid combination and interferon gamma (IFNγ) and CD40L-plasmid combination showed 45.5% and 54.5% complete remission (CR), respectively, on day 60; alternate treatments with both the plasmid combinations elicited 66.7% CR, while the control animals died within 48 days. Immune status analysis revealed that the density of dendritic cells significantly increased in tumors, particularly after GM-CSF- and CD40L-gene transfection, while that of regulatory T cells significantly decreased. The proportion of activated killer cells and antitumoral macrophages significantly increased, specifically after IFNγ and CD40L transfection. Furthermore, the level of the immune escape molecule programmed death ligand-1 decreased in tumors after transfecting these cytokine genes. As a result, tumor cell-specific transfection of these cytokine genes by the synthetic vehicle significantly promotes antitumor immune responses in the TME, a key aim for visceral tumor therapy.

Macrophage Mediated Abscopal Effects of Radiation Therapy

Radiation therapy (RT) can activate both innate and adaptive immune responses, and the combination of RT and immunotherapy is thought to be a promising strategy. We found that the "don't-eat-me" molecule CD47 is highly expressed on the surface of small cell lung cancer (SCLC) cells, a highly metastatic form of lung cancer, and that blockade of CD47 can enhance the phagocytosis of SCLC cells by macrophages. In this study, we investigated whether combining CD47 blockade and RT could synergize to inhibit the tumor growth of SCLC as well as other cancers in preclinical models. We evaluated efficacy and immune responses by RT and CD47 blockade in SCLC preclinical models as well as in colon cancer and lymphoma preclinical models. We engrafted cancer cells into both flanks of recipient mice and irradiated only one side of tumors with or without CD47 antibodies to investigate local and systemic effects of RT and CD47 blockade. We also used liver metastases models and endogenous lung tumor models to investigate anti-tumor effects and immune responses in their tumor microenvironments. In human patients, we investigated immune responses after RT using CIBERSORTx. We found CD47 blockade potently enhances the local antitumor effects of RT in all the pre-clinical models of SCLC we tested. Strikingly, CD47 blockade also stimulates systemic "abscopal" effects inhibiting non-irradiated SCLC tumors in mice receiving RT. These effects are observed in liver metastases models and endogenous lung tumor models as well as subcutaneous tumor models. Surprisingly, these abscopal effects are independent of T cells but require macrophages that migrate into non-irradiated tumor sites in response to inflammatory signals produced by RT and are locally activated by CD47 blockade to phagocytose cancer cells. Similar abscopal antitumor effects were observed in other cancer models treated with RT and CD47 blockade. We also found that RT increases tumor infiltrating macrophages in human cancer patients. Interestingly, these abscopal antitumor effects are enhanced by PD-1 blockade in some models. Our data clearly demonstrate macrophage-mediated abscopal responses of RT when combined with CD47 blockade in a range of cancer models. The systemic activation of antitumor macrophages following radiation and CD47 blockade may be particularly important in cancer patients who suffer from metastatic disease. RT is a part of standard-of-care and CD47-blocking strategies are in clinical trials, therefore our observations may be rapidly translatable to cancer patients.

Tumor microenvironment-activated, immunomodulatory nanosheets loaded with copper(II) and 5-FU for synergistic chemodynamic therapy and chemotherapy

The tumor microenvironment (TME) has a redox state that differs greatly from normal tissues, as characterized by the overexpression of H2O2 and glutathione (GSH). To address the GSH-related restrictions on chemodynamic therapy (CDT) efficacy, we have developed a Cu(II)-based CDT strategy. In this study, a novel organic–inorganic hybrid drug delivery system (LDH/HA/5-FU) was conceived and prepared by the intercalation of 5-FU into the interlayer of copper–aluminum layered double hydroxide (CuAl-LDH) via ion exchange strategy and the adsorption of hyaluronic acid (HA) on the surface of CuAl-LDH. Taking advantage of the pH-degradable property of CuAl-LDH and the CD44-targeting property of HA, the formed LDH/HA/5-FU nanosheets could specifically target tumor cells' overexpressing CD44 receptor, rapidly release Cu(II) and 5-FU in tumor cells, inducing tumor cell apoptosis and cuproptosis, and long-term intracellular GSH depletion and toxic hydroxyl radicals (·OH) generation could be achieved through the cyclic catalytic reaction of Cu(I)/Cu(II). Meanwhile, peritumoral injection of LDH/HA/5-FU nanosheets might function as an adjuvant to increase the levels of antitumor tumor-associated macrophages (TAMs) and T cells. In vivo experiments further verified that the intelligently designed LDH/HA/5-FU nanosheets successfully promoted the immune systems, with an excellent inhibition efficacy towards tumors by combining Cu-based CDT and chemotherapy, showing promising potential for solid tumor treatments.

Hepatocellular Carcinoma and Immunotherapy

With over 750,000 new cases identified annually, hepatocellular carcinoma (HCC) continues to be a threat, with current immunotherapies producing ineffective results in a majority of patients. PD1/PD-L1 antibodies have risen as another treatment option, but previous studies have only analyzed these antibodies in the tumor microenvironment. In order to understand how exactly these PD1/PD-L1 blockers affect macrophages and their anti-tumor macrophages, this study isolated the macrophage cells and tumor cells. Two protocols were followed, one with the B16F10 melanoma cell line, in order to determine proper procedures and determine a point of comparison, and one with the R1LWT liver cancer cell line. In the end, the B16F10 cell line responded positively to the PD1/PD-L1 antibodies, with increased MHCI/MHCII activation. On the other hand, the R1LWT cell line reacted oppositely, opening new inquiries into other pathways by which immunosuppression occurs.

Targeting MerTK decreases efferocytosis and increases anti-tumor immune infiltrate in prostate cancer

The prostate cancer tumor microenvironment (TME) is comprised of many cell types that can contribute to and influence tumor progression. Some of the most abundant prostate cancer TME cells are macrophages, which can be modeled on a continuous spectrum of M1-like (anti-tumor macrophages) to M2-like (pro-tumor macrophages). A function of M2-like macrophages is efferocytosis, the phagocytosis of apoptotic cells. Based on literature from other models and contexts, efferocytosis further supports the M2-like macrophage phenotype. MerTK is a receptor tyrosine kinase that mediates efferocytosis by binding phosphatidylserine on apoptotic cells. We hypothesize efferocytosis in the prostate cancer TME is a tumor-promoting function of macrophages and that targeting MerTK-mediated efferocytosis will slow prostate cancer growth and promote an anti-tumor immune infiltrate. The aims of this study are to measure efferocytosis of prostate cancer cells by in vitro human M1/M2 macrophage models and assess changes in the M2-like, pro-tumor macrophage phenotype following prostate cancer efferocytosis. Additionally, this study aims to demonstrate that targeting MerTK decreases prostate cancer efferocytosis and promotes an anti-tumor immune infiltrate. We have developed methodology using flow cytometry to quantify efferocytosis of human prostate cancer cells using the LNCaP cell line. We observed that M2 macrophages efferocytose the LNCaP cell line more than M1 macrophages. Following efferocytosis of LNCaP cells by M2 human monocyte-derived macrophages (HMDMs), we observed an increase in the M2-like, pro-tumor phenotype by flow cytometry cell surface marker analysis. By qRT-PCR, flow cytometry, and Western blot, we detected greater MerTK expression in M2 than M1 macrophages. Targeting MerTK with antibody Mer590 decreased LNCaP efferocytosis by M2 HMDMs, establishing the role of MerTK in prostate cancer efferocytosis. In the prostate cancer mouse model hi-myc, Mertk KO increased anti-tumor immune infiltrate including CD8 T cells. These findings support targeting MerTK-mediated efferocytosis as a novel therapy for prostate cancer.

Transcription factor Zhx2 is a checkpoint that programs macrophage polarization and antitumor response.

Macrophages are usually educated to tumor-associated macrophages (TAMs) in cancer with pro-tumor functions by tumor microenvironment (TME) and TAM reprogramming has been proposed as a potential tumor immunotherapy strategy. We recently demonstrated the critical role of Zinc-fingers and homeoboxes 2 (Zhx2) in macrophages' metabolic programming. However, whether Zhx2 is responsible for macrophage polarization and TAMs reprogramming is largely unknown. Here, we show that Zhx2 controls macrophage polarization under the inflammatory stimulus and TME. Myeloid-specific deletion of Zhx2 suppresses LPS-induced proinflammatory polarization but promotes IL-4 and TME-induced anti-inflammatory and pro-tumoral phenotypes in murine liver tumor models. Factors in TME, especially lactate, markedly decrease the expression of Zhx2 in TAMs, leading to the switch of TAMs to pro-tumor phenotype and consequent cancer progression. Notably, reduced ZHX2 expression in TAM correlates with poor survival of HCC patients. Mechanistic studies reveal that Zhx2 associates with NF-κB p65 and binds to the Irf1 promoter, leading to transcriptional activation of Irf1 in macrophages. Zhx2 functions in maintaining macrophage polarization by regulating Irf1 transcription, which may be a potential target for macrophage-based cancer immunotherapy.