Pro-tumor Macrophages Research Articles

Abstract 2667: Mucin 5AC inhibition via intratracheal RNAi delivery as an effective alternative strategy for prevention and treatment of Kras mutant lung cancer

Abstract Lung cancer, including lung adenocarcinoma (LUAD), is the leading cause of cancer-related deaths in the United States. KRAS mutations are the most prevalent oncogenic driver alteration in human LUAD. We have previously shown that high expression of Mucin 5 AC (MUC5AC), a main airway secretory mucin, is significantly associated with poor prognosis. Mucins are essential for mucociliary clearance and the maintenance of airway homeostasis, however, their overproduction could negatively affect the lung immune microenvironment and has been shown to promote airway diseases such as chronic obstructive pulmonary disease (COPD) and lung cancer. Previously, we have shown that genetic deletion of MUC5AC in a murine model of K-ras mutant lung adenocarcinoma (KM-LUAD) impedes tumor development and reduces pro-tumor inflammatory and immune phenotypes. These findings indicate that MUC5AC is a potential druggable target against KM-LUAD. In this study, we sought a translational approach and inhibited MUC5AC in our K-rasG12D mutant mouse model of LUAD via RNA interference (RNAi). Six-week-old mice were given RNAi (5mg/kg) or vehicle (saline) via biweekly intratracheal administration for 8 weeks. This approach led to a significant decrease in tumor burden (40%) and a trending increase in total white blood cell counts, predominantly macrophages in the lung tumor microenvironment which was associated with phenotypic changes in macrophages characterized by reduced expression of Fizz1, and M2 pro-tumor macrophage marker. Additionally, RNAi-treated mice had reduced expression of Siglec-E, a mucin receptor and known inhibitor of the TLR4 endocytosis and promoter of NF-ĸB signaling, as well as a significant decrease in IL-6, a product of the NF-ĸB pathway and promoter of STAT3 signaling which we have previously shown to have an essential role in promotion of KM-LUAD. In summary, these findings elucidate a potential mechanism by which MUC5AC protects tumor cells from apoptosis and immune-mediated cytotoxic activity but also highlights how MUC5AC RNAi is an effective indirect preventive and therapeutic strategy in high-risk individuals (smokers with COPD) and patients with early stage KM-LUAD, respectively. Citation Format: Melvin Zarghooni, Maria T. Grimaldo, Avantika Krishna, Michael J. Clowers, Linda Phan, Yasmina H. Rezai, Arnav Gaitonde, Carlos Rodriguez Reyna, Walter Velasco Torrez, Erik Bush, Seyed Javad Moghaddam. Mucin 5AC inhibition via intratracheal RNAi delivery as an effective alternative strategy for prevention and treatment of Kras mutant lung cancer [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 2667.

Abstract 5552: Uncovering clinically significant tumor microenvironment interaction programs across diverse cancers

Abstract Background: Cell-cell interactions (CCI) within the tumor microenvironment (TME) play pivotal roles in various tumor behaviors, including cancer growth, metastasis, immune evasion, and resistance to therapy. Compared to front-line treatments, targeting specific CCIs marks a paradigm shift in cancer therapy, aiming for enhanced response rates with reduced side effects. Although current approaches targeting immune regulation pathways exhibit effective anti-tumor responses, substantial variability in response rates across patients and cancer types persists. Consequently, a critical imperative emerges to discover novel interaction programs (IPs) across diverse TMEs, demonstrating clear clinical impact across a broad spectrum of patients. Methods: Characterization of pan cancer IPs. We compiled a pan cancer single-cell RNA-seq (scRNA-seq) atlas, including 4 million cells from 890 tumors across 14 cancer types. In each sample, CCIs were inferred using a ligand-receptor (LR) analysis framework, generating ranked CCI scores based on consensus from various LR inference methods. To find IPs enriched across TMEs, we applied an unsupervised factorization approach. The outcome represents CCIs as factors, where sample loading indicates factors’ strength, and feature loading highlights the cells and LR pairs constituting each IP. Evaluating IPs in clinical RNA-seq cohorts. Given the more immediate translational potential of bulk cancer sequencing for patient stratification, we devised an approach to predict IPs' strengths in bulk RNA-seq. Random Forest regression models using pseudo-bulk expression of cancer genes were trained to predict the loading (strength) of factors (IPs) in each sample in the scRNA-seq pan-cancer atlas. These models were subsequently applied to cancer samples in The Cancer Genome Atlas (TCGA) cohort to assess the clinical impact of various IPs on patient mortality. Results: We assessed the impact of IPs on 5-year overall survival using Cox proportional hazard models. We identified multiple factors significantly associated with survival in at least 4 cancer types. Notably, a robust prognostic factor linked to the inclusion of anti-tumor natural killer cell interactions and exclusion of pro-tumor macrophage interactions was found in MESO, LGG, ACC, and UVM (HR 1.6-6.6, p < 0.05). The validation of IPs using spatial transcriptomics and evaluation of IP prediction's utility in targeted treatment cohorts are currently underway. Conclusion: We highlight a potent method for detecting clinically significant IPs involving cells in TME communicating through LR pairs. The ability to assess these scRNA-seq-derived IPs in clinical bulk RNA-seq cohorts signifies a valuable advancement. Our approach holds promise for expanding the repertoire of cell therapies, identifying new treatment targets, and improving our predictive capabilities for responses to existing therapies. Citation Format: Ido Nofech-Mozes, Vivian Wang, Philip Awadalla, Sagi Abelson. Uncovering clinically significant tumor microenvironment interaction programs across diverse cancers [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 5552.

Abstract B075: Spatial genomics identifies cancer cell cytokines regulating ovarian cancer immunity

Abstract Ovarian cancer (OvCa) is among the most aggressive and lethal cancers with a poor response to immune checkpoint blockade (ICB) despite its modest tumor mutational burden and expression of known cancer antigens. Consequently, it is critical to understand the molecular mechanisms of tumor immune escape and resistance to immunotherapies. Intercellular signaling between cancer cells and the tumor microenvironment (TME) is a crucial determinant of tumor immunity and given the immunosuppressive nature of OvCa TME, it is of utmost importance to dissect how this immunosuppressed TME is established. To begin to determine which cancer-cell genes may function cell externally in controlling of OvCa immunity, we employed a first-of-its-kind spatial functional genomics approach we developed, called Perturb-map, which enables dozens of CRISPR gene knock-out (KO) cells to be resolved in a tissue/tumor by imaging, along with cells in the local TME. We used Perturb-map to determine how 35 different genes, identified through ligand-receptor analysis of ovarian tumors, influenced tumor response to anti-PD-1 treatment in an OvCa mouse model. Strikingly, Il4 KO tumors were unique amongst the 35 gene KOs making the tumors significantly more responsive to anti-PD-1 treatment despite not impacting tumor growth in the absence of immunotherapy. Further experiments with single CRISPR KO tumors validated the Perturb-map finding, where Il4 KO combined with anti-PD-1 treatment resulted in a major reduction in tumor growth and a significant increase in survival. Cyclic immunofluorescence (CyCIF) multiplex imaging revealed that loss of cancer cell-derived IL-4 led to a significant reduction in pro-tumor macrophages and an increase in dendritic cell (DC) subsets as well as both progenitor and terminally exhausted CD8 T cells. Upon anti-PD-1 treatment, Il4 KO tumors had significant enrichment for activated CD8 T cells and B cells. Importantly, spatial analysis of the tumor samples identified enrichment of T follicular helper cells, B cells, and DCs in tertiary lymphoid structures in tumors with combined IL-4 loss and PD-1 inhibition. Further, we analyzed a human OvCa single-cell transcriptomics dataset and demonstrated that an IL-4 response signature is expressed in tumor-associated macrophages, indicating active IL-4 signaling in human ovarian tumors. Importantly, we employed a CRISPR-knock-in expression reporter in mouse tumors as well as immunohistochemistry of both human and mouse tumor samples to confirm that IL-4 is expressed by cancer cells. Thus, we identified cancer cell-secreted IL-4 as a potent regulator of OvCa TME and response to ICB. Given that an anti-IL-4-receptor antibody is already in clinical use to treat immune diseases, our findings have the potential to be translated to the clinical management of this dreadful disease. Citation Format: Gurkan Mollaoglu, Alexander Tepper, Hunter Potak, Luisanna Pia, Angelo Amabile, Chiara Falcomata, Jaime Mateus-Tique, Noam Rabinovich, Rachel Brody, Lindsay Browning, Jia-Ren Lin, Peter Sorger, Sandro Santagata, Miriam Merad, Alessia Baccarini, Brian Brown. Spatial genomics identifies cancer cell cytokines regulating ovarian cancer immunity [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr B075.

INHBA is Enriched in HPV-negative Oropharyngeal Squamous Cell Carcinoma and Promotes Cancer Progression.

Patients with HPV-negative OPSCC have a poorer prognosis due to distinct molecular pathways. This study reveals significant transcriptomic differences between HPV-negative and HPV-positive OPSCC, identifying INHBA as a key upregulated gene in HPV-negative OPSCC's oncogenic pathways. INHBA is crucial in promoting EMT, cell proliferation, and an immunosuppressive tumor environment, suggesting its potential as a therapeutic target for HPV-negative OPSCC.

Targeting of M2 macrophages with IL-13-functionalized liposomal prednisolone inhibits melanoma angiogenesis in vivo

The intricate cooperation between cancer cells and nontumor stromal cells within melanoma microenvironment (MME) enables tumor progression and metastasis. We previously demonstrated that the interplay between tumor-associated macrophages (TAMs) and melanoma cells can be disrupted by using long-circulating liposomes (LCLs) encapsulating prednisolone phosphate (PLP) (LCL-PLP) that inhibited tumor angiogenesis coordinated by TAMs. In this study, our goal was to improve LCL specificity for protumor macrophages (M2-like (i.e., TAMs) macrophages) and to induce a more precise accumulation at tumor site by loading PLP into IL-13-conjugated liposomes (IL-13-LCL-PLP), since IL-13 receptor is overexpressed in this type of macrophages. The IL-13-LCL-PLP liposomal formulation was obtained by covalent attachment of thiolated IL-13 to maleimide-functionalized LCL-PLP. C57BL/6 mice bearing B16.F10 s.c melanoma tumors were used to investigate the antitumor action of LCL-PLP and IL-13-LCL-PLP. Our results showed that IL-13-LCL-PLP formulation remained stable in biological fluids after 24h and it was preferentially taken up by M2 polarized macrophages. IL-13-LCL-PLP induced strong tumor growth inhibition compared to nonfunctionalized LCL-PLP at the same dose, by altering TAMs-mediated angiogenesis and oxidative stress, limiting resistance to apoptosis and invasive features in MME. These findings suggest IL-13-LCL-PLP might become a promising delivery platform for chemotherapeutic agents in melanoma.

Abstract B047: Epigenetic reprogramming by CBP/P300 bromodomain inhibition of triple-negative breast cancer and the immune microenvironment

Abstract Triple-negative breast cancer (TNBC) is a biologically heterogeneous and clinically important breast cancer subtype because if considered a distinct disease, TNBC would rank as the 5th leading cause of cancer deaths in women. TNBC patients resistant to standard-of-care therapies have poor survival. Tumor-associated immune cells such as neutrophils and macrophages can display a pro-tumoral phenotype and contribute to therapy resistance. Epigenetic enzymes have been shown to reprogram both the tumor cells and tumor-associated immune cells. Thus, targeting epigenetic enzymes is a potential strategy to improve the response of TNBC to standard-of-care therapies. Previously, our laboratory performed an epigenetic drug screen and identified several epigenetic inhibitors. We selected IACS-70654, a novel selective p300/CBP BRD inhibitor, to be the primary focus of this study. We determined the effects of IACS-70654 in our unique syngeneic TNBC mouse models. In a neutrophil-enriched luminal-like TNBC model 2208L, IACS-70654 as a single agent initially caused tumor regression and then durably prevented tumor growth, which is not usually observed for single agents in these aggressive TNBC models. IACS-70654 also substantially decreased infiltrated neutrophils. In the peripheral blood and bone marrow of 2208L tumor-bearing mice, IACS-70654 reduced neutrophils to the same level as non-tumor-bearing mice. Based on this finding, we treated 2208L tumors with IACS-70654 in combination with immune checkpoint blockade anti-PD1 and docetaxel. After 27 days, all treatments besides anti-PD1 were stopped, and the combination-treated tumors grew slower than the ones only treated with IACS-70654. More importantly, in tumors treated with IACS-70654 in combination with anti-PD1, there was a substantial increase in CD8+ T cell infiltration. In addition, B cell infiltration was induced in the tumor stroma, indicating that IACS-70654 stimulated immune response in the tumor microenvironment. In contrast, IACS-70654 failed to significantly reduce the growth of T12, a Claudin-low macrophage-enriched model. Therefore, we hypothesized that the infiltrated immune profiles, specifically myeloid cell infiltration, might partially account for this differential response. Accordingly, we studied the effects of IACS-70654 in two additional models, basal-like T6 and luminal-like 2250L tumors, which also have relatively high neutrophil infiltration. However, one of the principal differences in immune profiles between the two models and 2208L is that they also are infiltrated with twice as many macrophages. The response of infiltrated immune cells to IACS-70654 in those two models differed from those of 2208L tumors. IACS-70654 reduced monocytic myeloid-derived suppressor cells and pro-tumor macrophages. These results illustrate the immune heterogeneity among our tumor models. We are currently performing ChIP-seq and single-cell ATAC-seq to elucidate potential mechanisms and pathways employed by IACS-70654 in both tumor and tumor-associated immune cells. Citation Format: Xueying Yuan, Michael Soth, Na Zhao, Philip Jones, Jeffrey Rosen. Epigenetic reprogramming by CBP/P300 bromodomain inhibition of triple-negative breast cancer and the immune microenvironment [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr B047.

IL4 receptor targeting enables nab-paclitaxel to enhance reprogramming of M2-type macrophages into M1-like phenotype via ROS-HMGB1-TLR4 axis and inhibition of tumor growth and metastasis.

Rationale: Nab-paclitaxel (Abx) is widely employed in malignant tumor therapy. In tumor cells and pro-tumoral M2-type macrophages, the IL4 receptor (IL4R) is upregulated. This study aimed to elucidate the selective delivery of Abx to M2-type macrophages by targeting IL4R and reprogramming them into an anti-tumoral M1-type. Methods: Abx was conjugated with the IL4R-binding IL4RPep-1 peptide using click chemistry (IL4R-Abx). Cellular internalization, macrophage reprogramming and signal pathways, and tumor growth and metastasis by IL4R-Abx were examined. Results: IL4R-Abx was internalized into M2 macrophages more efficiently compared to the unmodified Abx and control peptide-conjugated Abx (Ctrl-Abx), which was primarily inhibited using an anti-IL4R antibody and a receptor-mediated endocytosis inhibitor compared with a macropinocytosis inhibitor. IL4R-Abx reprogrammed the M2-type macrophages into M1-like phenotype and increased reactive oxygen species (ROS) levels and extracellular release of high mobility group box 1 (HMGB1) in M2 macrophages at higher levels than Abx and Ctrl-Abx. The conditioned medium of IL4R-Abx-treated M2 macrophages skewed M2 macrophages into the M1-like phenotype, in which an anti-HMGB1 antibody and a toll-like receptor 4 (TLR4) inhibitor induced a blockade. IL4R-Abx accumulated at tumors, heightened immune-stimulatory cells while reducing immune-suppressing cells, and hampered tumor growth and metastasis in mice more efficiently than Abx and Ctrl-Abx. Conclusions: These results indicate that IL4R-targeting allows enhancement of M2-macrophage shaping into M1-like phenotype by Abx through the ROS-HMGB1-TLR4 axis, improvement of antitumor immunity, and thereby inhibition of tumor growth and metastasis, presenting a new approach to cancer immunotherapy.

Commentary: PFKFB3 overexpression in monocytes of patients with colon but not rectal cancer programs pro-tumor macrophages and is indicative for higher risk of tumor relapse.

Commentary: PFKFB3 overexpression in monocytes of patients with colon but not rectal cancer programs pro-tumor macrophages and is indicative for higher risk of tumor relapse.

SNHG17 alters anaerobic glycolysis by resetting phosphorylation modification of PGK1 to foster pro-tumor macrophage formation in pancreatic ductal adenocarcinoma

BackgroundWithin the tumor immune microenvironment (TME), tumor-associated macrophages (TAMs) are crucial in modulating polarization states to influence cancer development through metabolic reprogramming. While long non-coding RNAs (lncRNAs) have been shown to play a pivotal role in the progression of various cancers, the underlying mechanisms by which lncRNAs alter M2 polarization through macrophage metabolism remodeling remain unelucidated.MethodsRNA sequencing was used to screen for differentially expressed lncRNAs in TAMs and normal tissue-resident macrophages (NTRMs) isolated from pancreatic ductal adenocarcinoma (PDAC) tissues, whilst RT-qPCR and FISH were employed to detect the expression level of SNHG17. Moreover, a series of in vivo and in vitro experiments were conducted to assess the functions of SNHG17 from TAMs in the polarization and glycolysis of M2-like macrophages and in the proliferation and metastasis of pancreatic cancer cells (PCs). Furthermore, Western blotting, RNA pull-down, mass spectrometry, RIP, and dual-luciferase assays were utilized to explore the underlying mechanism through which SNHG17 induces pro-tumor macrophage formation.ResultsSNHG17 was substantially enriched in TAMs and was positively correlated with a worse prognosis in PDAC. Meanwhile, functional assays determined that SNHG17 promoted the malignant progression of PCs by enhancing M2 macrophage polarization and anaerobic glycolysis. Mechanistically, SNHG17 could sponge miR-628-5p to release PGK1 mRNA and concurrently interact with the PGK1 protein, activating the pro-tumorigenic function of PGK1 by enhancing phosphorylation at the T168A site of PGK1 through ERK1/2 recruitment. Lastly, SNHG17 knockdown could reverse the polarization status of macrophages in PDAC.ConclusionsThe present study illustrated the essential role of SNHG17 and its molecular mechanism in TAMs derived from PDAC, indicating that SNHG17 might be a viable target for PDAC immunotherapy.

RALDH1 Inhibition Shows Immunotherapeutic Efficacy in Hepatocellular Carcinoma.

Globally, hepatocellular carcinoma (HCC) is one of the most commonly diagnosed cancers and a leading cause of cancer-related death. We previously identified an immune evasion pathway whereby tumor cells produce retinoic acid (RA) to promote differentiation of intratumoral monocytes into protumor macrophages. Retinaldehyde dehydrogenase 1 (RALDH1), RALDH2, and RALDH3 are the three isozymes that catalyze RA biosynthesis. In this study, we have identified RALDH1 as the key driver of RA production in HCC and demonstrated the efficacy of RALDH1-selective inhibitors (Raldh1-INH) in suppressing RA production by HCC cells. Raldh1-INH restrained tumor growth in multiple mouse models of HCC by reducing the number and tumor-supporting functions of intratumoral macrophages as well as increasing T-cell infiltration and activation within tumors. Raldh1-INH also displayed favorable pharmacokinetic, pharmacodynamic, and toxicity profiles in mice thereby establishing them as promising new drug candidates for HCC immunotherapy.

Abstract B014: Spatial genomics identifies cancer cell cytokines regulating ovarian cancer immunity

Abstract Ovarian cancer (OvCa) is among the most aggressive and lethal cancers with a poor response to immune checkpoint blockade (ICB) despite its modest tumor mutational burden and expression of known cancer antigens. Consequently, it is critical to understand the molecular mechanisms of tumor immune escape and resistance to immunotherapies. Intercellular signaling between cancer cells and the tumor microenvironment (TME) is a crucial determinant of tumor immunity and given the immunosuppressive nature of OvCa TME, it is of utmost importance to dissect how this immunosuppressed TME is established. To begin to determine which cancer-cell genes may function cell externally in controlling of OvCa immunity, we employed a first-of-its-kind spatial functional genomics approach we developed, called Perturb-map, which enables dozens of CRISPR gene knock-out (KO) cells to be resolved in a tissue/tumor by imaging, along with cells in the local TME. We used Perturb-map to determine how 35 different genes, identified through ligand-receptor analysis of ovarian tumors, influenced tumor response to anti-PD-1 treatment in an OvCa mouse model. Strikingly, Il4 KO tumors were unique amongst the 35 gene KOs making the tumors significantly more responsive to anti-PD-1 treatment despite not impacting tumor growth in the absence of immunotherapy. Further experiments with single CRISPR KO tumors validated the Perturb-map finding, where Il4 KO combined with anti-PD-1 treatment resulted in a major reduction in tumor growth and a significant increase in survival. Cyclic immunofluorescence (CyCIF) multiplex imaging revealed that loss of cancer cell-derived IL-4 led to a significant reduction in pro-tumor macrophages and an increase in dendritic cell (DC) subsets as well as both progenitor and terminally exhausted CD8 T cells. Upon anti-PD-1 treatment, Il4 KO tumors had significant enrichment for activated CD8 T cells and B cells. Importantly, spatial analysis of the tumor samples identified enrichment of T follicular helper cells, B cells, and DCs in tertiary lymphoid structures in tumors with combined IL-4 loss and PD-1 inhibition. Further, we analyzed a human OvCa single-cell transcriptomics dataset and demonstrated that an IL-4 response signature is expressed in tumor-associated macrophages, indicating active IL-4 signaling in human ovarian tumors. Importantly, we employed a CRISPR-knock-in expression reporter in mouse tumors as well as immunohistochemistry of both human and mouse tumor samples to confirm that IL-4 is expressed by cancer cells. Thus, we identified cancer cell-secreted IL-4 as a potent regulator of OvCa TME and response to ICB. Given that an anti-IL-4-receptor antibody is already in clinical use to treat immune diseases, our findings have the potential to be translated to the clinical management of this dreadful disease. Citation Format: Gurkan Mollaoglu, Alexander Tepper, Hunter Potak, Luisanna Pia, Angelo Amabile, Chiara Falcomata, Jaime Mateus-Tique, Noam Rabinovich, Rachel Brody, Lindsay Browning, Jia-Ren Lin, Peter Sorger, Sandro Santagata, Miriam Merad, Alessia Baccarini, Brian Brown. Spatial genomics identifies cancer cell cytokines regulating ovarian cancer immunity [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 B014.

Green Nanotechnology of Yucca filamentosa- Phytochemicals-Functionalized Gold Nanoparticles-Antitumor Efficacy Against Prostate and Breast Cancers.

We report an innovative green nanotechnology utilizing an electron-rich cocktail of phytochemicals from Yucca filamentosa L. to synthesize biocompatible gold nanoparticles without the use of any external chemical reducing agents and evaluate their anti-cancer activity. Yucca filamentosa L. extract, containing a cocktail of phytochemicals, was prepared, and used to transform gold salt into Y. filamentosa phytochemicals encapsulated gold nanoparticles (YF-AuNPs). Additionally, gum arabic stabilized YF-AuNPs (GAYF-AuNPs) were also prepared to enhance the in vitro/in vivo stability. Anticancer activity was evaluated against prostate (PC-3) and breast (MDAMB-231) cancer cell lines. Targeting abilities of gold nanoparticles were tested using pro-tumor macrophage cell lines. Comprehensive characterization of new nanomedicine agents YF-AuNPs and GAYF-AuNPs revealed spherical, and monodisperse AuNPs with moderate zeta potentials (-19 and -20 mV, respectively), indicating in vitro/in vivo stability. The core size of YF-AuNPs (14 ± 5 nm) and GAYF-AuNPs (10 ± 5 nm) is suitable for optimal penetration into tumor cells through both enhanced permeability and retention (EPR) effect as well as through the receptor mediated endocytosis. Notably, YF-AuNPs exhibited potent anticancer activity against prostate (PC-3) and breast tumors (MDAMB-231) by inducing early and late apoptotic stages. Moreover, YF-AuNPs resulted in elevated levels of anti-tumor cytokines (TNF-α and IL-12) and reduced levels of pro-tumor cytokines (IL-6 and IL-10), provide compelling evidence on the immunomodulatory property of YF-AuNPs. Overall, these Y. filamentosa phytochemicals functionalized nano-Ayurvedic medicine agents demonstrated selective toxicity to cancer cells while sparing normal cells. Most notably, to our knowledge, this is the first study that shows YF-AuNP's targeting efficacy toward pro-tumor macrophage cell lines, suggesting an immunomodulatory pathway for cancer treatment. This work introduces a novel avenue for herbal and nano-Ayurvedic approaches to human cancer treatment, mediated through selective efficacy and immunomodulatory potential.

Addition of Acalabrutinib to Lenalidomide and Rituximab Induces High Complete Response Rates in Patients with Previously Untreated Follicular Lymphoma: Results of a Phase II Study

Background. We previously reported pro-tumoral macrophages may be associated with resistance to lenalidomide and rituximab (R 2) treatment in follicular lymphoma (FL) ( Marques-Piubelli, Blood Adv 2022). As pre-clinical studies suggest that BTK inhibition can mitigate the crosstalk between monocyte/macrophages and FL B cells, we hypothesized that acalabrutinib, a specific BTK inhibitor, may synergize with R 2 without increasing toxicity. In agreement with this, the combination of acalabrutinib and R 2 (aR 2) was shown to be safe and effective in patients with relapsed FL ( Strati P et al, ASH 2022). Here, we investigated the safety and efficacy of aR 2 in previously untreated FL patients and analyzed the biological effects of this combination on peripheral blood immune cells. Methods. This phase 2 single arm study (NCT04404088) was conducted between 09/2020 and 09/2021 (data cutoff 06/2023). Adult patients with previously untreated FL, grades 1 to 3A, stage 3-4, and meeting indication for treatment per GELF criteria were included. Treatment consisted of acalabrutinib lead-in at 100 mg PO twice a day for one 28-day cycle and continued for total of 13 cycles, lenalidomide 20 mg PO daily on days 1-21, during cycle 2-13, and rituximab 375 mg/m 2 IV weekly during cycle 2, and on day 1 of cycle 3-13. The primary endpoint was best complete response (CR) rate per Lugano 2014 criteria. Bulk RNA sequencing with deconvolution to characterize circulating immune cells was performed on peripheral blood collected at baseline, and on day 1 of cycles 2 and 6. Results. We enrolled and treated 24 patients. Baseline characteristics are shown in the Table. Median number of cycles was 13 (range, 6-13) and 15 (62.5%) patients experienced a cycle delay, due to COVID19 in 11 (46%) cases; 6 (25%) patients required dose reduction of lenalidomide, but none discontinued, and 2 (8%) required dose reduction of acalabrutinib and 1 discontinued. The most common (>5% of patients) grade 3-4 adverse events were neutropenia (58%), liver function test elevation (17%), infection (12.5%; 2 out of 3 related to COVID19), anemia (8%) and skin rash (8%). Best ORR was 100%, best CR rate was 92%, and median time to CR was 3 months ( Figure). After a median follow-up of 26.8 months (95% CI 24.6-29.6 months), 6 (25%) patients had disease progression, including 2 who transformed at 5 and 7 months. The 2-year PFS rate was 79.2% (95% CI, 64.5-97.2%). At data cutoff, 2 patients have died, 1 due to COVID19 (while in CR, at 14 months) and 1 due to transformed lymphoma (at 10 months). The 2-year OS rate was 91.7% (95% CI, 81.3-100%). While the frequency of circulating immune cells were unchanged after single agent acalabrutinib, its use associated with a significant decrease in the expression of VMO1 (FDR 0.001), a gene encoding for a protein expressed on extra-cellular exosomes derived from non-classical monocytes, and a significant increase in gene signatures of monocyte proliferation (p=0.01), TNF response (p=0.01), anti-viral (p=0.006) and anti-tumoral activity (p=0.009), consistent with the expected effect of BTK inhibition in monocytes. After 5 cycles of aR 2, we observed a decrease in the frequency of circulating B cells (p=0.003) and regulatory NK cells (p=0.01), and an increase in the frequency of circulating naive T cells (p=0.004), CD4+ T cells (p=0.005) and classical monocytes (p=0.003). Conclusion. Our results indicate that the addition of acalabrutinib to R 2 is a safe and effective frontline non-chemotherapy regimen for FL patients, resulting in high CR rates, and induces favorable biological changes in multiple circulating immune cells. Since CRs occurred early, the study has been amended to include 26 additional patients who will be treated with only 6 cycles of aR2.

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.

EXTH-44. ENHANCING GLIOBLASTOMA CLEARANCE: EMPOWERING ANTI-EGFRVIII CAR T CELLS WITH A PARACRINE SIRPΓ-DERIVED CD47 BLOCKER

Abstract A major challenge for chimeric antigen receptor (CAR) T cell therapy against glioblastoma (GBM) is the immunosuppressive microenvironment (iTME), which is densely populated by protumoral glioma-associated microglia and macrophages (GAMs). Blockade of CD47, a “don’t-eat-me” signal overexpressed by GBM cells, disrupts the CD47-SIRPα axis, and regulates the phagocytic function of GAMs. However, systemic anti-CD47 monotherapy has been ineffective against human solid tumors to date. Here, we combined local CAR T therapy with paracrine GAM modulation to synergistically eliminate GBM. We engineered an armored CAR T cell against EGFRvIII that constitutively secretes a SIRPγ-related protein (SGRP) with high affinity to CD47. Anti-EGFRvIII-SGRP CAR T cells eliminated EGFRvIII+ GBM in vitro, and eradicated EGFRvIII-heterogeneous (EGFRvIII+ and EGFRvIII- mosaic) orthotopically xenografted tumors by locoregional application in vivo. This resulted in significant tumor-free long-term survival, followed by partial tumor control upon re-challenge. Combination of anti-CD47 antibodies with anti-EGFRvIII-CAR T cells failed to achieve synergy, pointing towards the importance of sustained paracrine GAM conversion. Paracrine SGRP enhanced CAR T infiltration, tumor clearance and GAM reconversion to an antitumor phenotype as shown by multidimensional brain immunofluorescence microscopy and in-depth spectral flow cytometry multiplexing. Moreover, the plasma of anti-EGFRvIII-SGRP CAR T-treated mice displayed a distinct signature of innate immune activation including TNFα and CCL3. Validation of the anti-EGFRvIII-SGRP CAR T in human explants is ongoing. Furthermore, armoring anti-CD19 CAR T cells with SGRP resulted in superior efficacy in a peripheral lymphoma mouse model compared to anti-CD19 CAR T treatment alone, most problably due to innate immune activation. Thus, local anti-EGFRvIII-SGRP CAR T cell therapy combines the potent antitumor effect of engineered T cells with the modulation of the surrounding innate iTME, inducing a synergistic clearance of GBM in a manner that overcomes known mechanisms of CAR T therapy evasion, such as tumor immune suppression and antigen escape.

IMMU-04. LOW-INTENSITY PULSED ULTRASOUND ENHANCES CAR T CELL THERAPY BY INDUCING TWO-WAY IMMUNE CELL TRAFFICKING – T CELL INTO TUMOR AND MYELOID CELL INTO PERIPHERY IN A SYNGENEIC MOUSE GLIOBLASTOMA MODEL

Abstract Chimeric Antigen Receptor (CAR) T-cell therapy has been successful against hematological malignancies. However, it has not established efficacy in glioblastoma (GBM) patients. Significant challenges currently limiting the effectiveness of CAR T-cell therapy for patients with GBM include poor CAR-T cell trafficking to GBM due to the blood-brain barrier, and the presence of highly immunosuppressive and pro-tumoral tumor-associated macrophages (TAMs) in the tumor microenvironment. Furthermore, the heterogeneous expression of target antigens in GBM can lead to antigen escape and therapeutic resistance. Low-intensity pulsed ultrasound with microbubbles (LIPU/MB) has been shown to enhance CAR T-cell trafficking and improve survival in immunocompromised xenograft mice GBM models. In this study, we investigated whether multiple rounds of LIPU/MB can improve not only CAR T-cell but also endogenous T-cell infiltration into the tumors and induce myeloid cell trafficking into lymph nodes (LNs), thus enhancing CAR T-cell therapy against GBM in the immunocompetent syngeneic mice model. We recently developed a novel immunocompetent transgenic (Tg) mouse that expresses an anti-EGFRvIII CAR on T-cells and enables us to investigate the interaction between CAR T-cells and the endogenous immune environment. C57BL6/J mice bearing intracerebral EGFRvIII+ GBM received an intravenous administration of anti-EGFRvIII CAR T-cells derived from Tg-mice. The mice were stratified to receive single-LIPU (1 min before CAR T-cells administration) or triple-LIPU/MB (1 min before CAR T-cells administration, then two and four days later). Triple-LIPU/MB significantly suppressed tumor growth and improved survival and was associated with increased CAR T-cells and endogenous T-cells infiltration into the tumors compared with single-LIPU/MB or without LIPU. Furthermore, draining cervical LNs showed increased macrophages trafficking after triple-LIPU/MB. Moreover, TAMs isolated from triple-LIPU/MB mice brains showed higher levels of MHC-II expression. These results suggest that LIPU/MB may promote the tumor antigen presentation to endogenous T-cells and enhance the efficacy of CAR T-cell therapy for GBM.

Targeting and repolarizing M2-like tumor-associated macrophage-mediated MR imaging and tumor immunotherapy by biomimetic nanoparticles

Anti-tumor M1-like and pro-tumor M2-like tumor-associated macrophages (TAMs) coexist in tumor microenvironments (TME). The adverse effects of these M1/M2 subsets on tumors directly affect the current strategies to improve anti-tumor immune response. Therefore, it has attracted great attention to change the tumor immunosuppressive microenvironment by reprogramming TAMs. In this paper, we constructed biomimetic nanoparticles (HMMDN-Met@PM) targeting M2-like TAMs for macrophage re-polarization. In detail, the core of the biomimetic nanoparticles is metformin-loaded hollow mesoporous manganese dioxide nanoparticles (HMMDN-Met). Benefited from the hollow and porous structure of HMMDN, metformin, the regulator of M1/M2 adopted in this work, can be easily and widely loaded into HMMDN. Moreover, macrophage membranes were utilized for HMMDN-Met coating (HMMDN-Met@MM) to prevent the premature drug leakage and provide specific molecular recognition/TME targeting. In addition, M2 macrophage targeting peptide (M2pep) was modified on the surface of macrophage membrane to specifically deliver the drug to M2-like TAMs to promote the polarization of M2 to M1 macrophages. Through in vitro and in vivo studies, we found that the expression of surface markers and inflammatory factors CD206, Arg-1 and IL-10 of type M2 macrophages decreased, while the surface markers of type M1 macrophages and the expression of inflammatory factors CD80, TNF-α and iNOS increased, indicating the successful re-polarization of M2 macrophages and finally realizing the inhibition of tumor growth. At the same time, under the acidic and GSH conditions of tumor, HMMDN was decomposed into Mn2+, which is a contrast agent for magnetic resonance imaging, thus realizing the tracking of tumor. This work practices biomimetic nanosystem in targeted imaging and immunotherapy, paving the way for strategy designing for tumor inhibition.

Oxidative stress is involved in immunosuppression and macrophage regulation in glioblastoma

Oxidative stress dually affected cancer progression, while its effect on glioblastomas remained unclear. Herein, we clustered the multicenter glioblastoma cohorts based on the oxidative-stress-responsive genes (OSS) expression. We found that cluster 2 with high OSS levels suffered a worse prognosis. Functional analyses and immune-related analyses results exhibited that M2-like pro-tumoral macrophages and neutrophils were enriched in cluster 2, while Natural killer cells' infiltration was decreased. The increased M2-like pro-tumoral macrophages in cluster 2 was confirmed by immunofluorescence. An integrated single-cell analysis validated the malignant features of cluster 2 neoplastic cells and discovered their crosstalk with M2-like pro-tumoral macrophages. Moreover, we observed that SOD3 knockdown might decrease the M2-like pro-tumoral transformation of macrophage in vitro and in vivo. Comprehensively, we revealed oxidative stress' prognostic and immunosuppressive potential in glioblastoma and discovered SOD3's potential role in regulating macrophage M2-like pro-tumoral transformation.

Emodin reduces surgical wounding-accelerated tumor growth and metastasis via macrophage suppression in a murine triple-negative breast cancer model.

It has been suspected that tumor resection surgery itself may accelerate breast cancer (BC) lung metastasis in some patients. Emodin, a natural anthraquinone found in the roots and rhizomes of various plants, exhibits anticancer activity. We examined the perioperative use of emodin in our established surgery wounding murine BC model. Emodin reduced primary BC tumor growth and metastasis in the lungs in both sham and surgical wounded mice, consistent with a reduction in proliferation and enhanced apoptosis (primary tumor and lungs). Further, emodin reduced systemic inflammation, most notably the number of monocytes in the peripheral blood and reduced pro-tumoral M2 macrophages in the primary tumor and the lungs. Consistently, we show that emodin reduces gene expression of select macrophage markers and associated cytokines in the primary tumor and lungs of wounded mice. Overall, we demonstrate that emodin is beneficial in mitigating surgical wounding accelerated lung metastasis in a model of triple-negative BC, which appears to be mediated, at least in part, by its actions on macrophages. These data support the development of emodin as a safe, low-cost, and effective agent to be used perioperatively to alleviate the surgery triggered inflammatory response and consequential metastasis of BC to the lungs.

Functional impact of oxygen-saturated zeolite nanoparticles on macrophages in the context of glioblastoma: an in vitro and in vivo study.

In the context of glioblastoma (GBM), hypoxia and inflammation are two main players of the tumor microenvironment. Hypoxia stimulates various features involves in tumor growth and also maintains a specific environment that favors protumor macrophages. Therefore, targeting hypoxia could potentially restore an anti-tumor M1 phenotype in macrophages. Besides, iron demonstrated its capacity to stimulate the polarization of macrophages towards an M1-like phenotype. In this paper we took advantages of microporous nanoparticles to co-deliver both oxygen and iron to bone marrow derived macrophages (BMDM) enabling the investigation of changes in polarization status and proteomic profiles. The nanoparticles were used in two in vivo models of glioblastoma, specifically, in both immunodeficient and immunocompetent settings. Our in vitro findings revealed that iron doped nanoparticles, saturated with oxygen were deemed safe for macrophages but did not demonstrate the capacity to change the M1 or M2 phenotypes. However, these nanoparticles induced some changes in proteomics pathways. The present study reports on in vivo experimentation that revealed the effects of nanoparticles on the hypoxic fraction, tumor volume, and macrophage phenotype in a GBM model. The findings indicated that the presence of nanoparticles led to a reduction in the hypoxic fraction in one of the GBM models, while no significant changes were observed in the tumor volume or macrophage phenotype. The present data showed that nanoparticles possess the capability of delivering both oxygen and iron to macrophages; though, they do not possess the ability to effectively repolarize M2 macrophages. Such strategies could be used in conjunction with other potent molecules to avoid M1 macrophages to inevitably differentiate to M2 macrophages.