Immunosuppressive M2 Macrophages Research Articles

Abstract 5117: Combined treatment with the epigenetic drug CM272 and an anti-BCL-XL proapoptotic drug sensitizes solid tumors to immune checkpoint blockade

Abstract Epigenetic modulators in combination with proapoptotic drugs have become a standard of treatment for acute myeloid leukemia. However, the clinical efficacy of these combinations in solid tumors has shown to be negligible. Our group has demonstrated that the epigenetic drug CM272, which targets DNMT and G9a, induces synergistic responses in combination with proapoptotic drugs in hematological malignancies (Cancer Discovery 2021). We have also reported that CM272 induces an immunogenic cell death that sensitizes tumor cells to immune checkpoint blockade (ICB) (Nature Medicine 2020). Here, we hypothesized that CM272 in combination with proapoptotic drugs may sensitize tumor cells to ICB, resulting in profound anti-tumor responses. To test this hypothesis, we evaluated in vitro the sensitivity of cancer cells to different combinations of epigenetic and proapoptotic drugs. The most efficient combination was tested in various immunocompetent mouse models, followed by an immunophenotypic characterization of the tumor microenvironment. We found that CM272 in combination with inhibitors of BCL-XL, BCL2 or MCL1, yielded synergistic in vitro responses in a large collection of human and mouse cell lines derived from solid tumors. Mechanistically, the treatments induced the expression of endogenous retroelements/retroviruses, which led to the activation of RIG-1 and MDA5 viral sensors, ATP hydrolysis and tumor cell death. Remarkably, we found that CM272 in combination with BCL-XL inhibition had more potent anti-tumor effects than that found with BCL2 or MCL1 inhibitors. In vivo, the triple combination CM272, A1331852 (BCL-XL inhibitor) and an anti-PD-1 moAb significantly reduced tumor growth and increased overall survival in three subcutaneous lung cancer models (LLC, 393P, Lacun-3) in comparison to double treatments. Moreover, this triple combination also induced significant anti-tumor responses in subcutaneous mouse models of colon cancer (MC38) and melanoma (B16), as well as in orthotopic models of lung cancer (LLC), glioblastoma (CT-2A) and breast cancer (ANV5), leading to prolonged survival and cure in a fraction of animals. The triple therapy was associated with a significant increase in the ratio of CD8 T versus immunosuppressive Treg cells, and M1 versus M2 macrophages. In conclusion, we report a novel regimen combining a dual epigenetic inhibitor, an anti-BCL-XL, and an anti-PD-1 moAb that results in potent responses in multiple pre-clinical models of solid tumors. The mechanisms underneath the antitumor responses include the modulation of the energy metabolism in tumor cells, leading to cell death boosted by an anti-BCL-XL pro-apoptotic drug, along with fostering the immune system to generate an efficient anti-tumor response assisted by ICB. This study reveals the potential of epigenetic therapeutics to treat and cure patients with solid tumors. Citation Format: Yaiza Senent, Vicente Fresquet, Victoria Jimenez, Karmele Valencia, Ana Remirez, Francisco Exposito, Marisol Gonzalez-Huarriz, Borja Ruiz-Fernandez de Cordoba, Haritz Moreno, Daniel Ajona, Marta M Alonso, Fernando Lecanda, Antonio Pineda-Lucena, Felipe Prosper, Alfonso Calvo, Jose Angel Martinez-Climent, Ruben Pio. Combined treatment with the epigenetic drug CM272 and an anti-BCL-XL proapoptotic drug sensitizes solid tumors to immune checkpoint blockade. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5117.

Vector Aided Microenvironment programming (VAMP): reprogramming the TME with MVA virus expressing IL-12 for effective antitumor activity

BackgroundTumor microenvironment (TME) represents a critical hurdle in cancer immunotherapy, given its ability to suppress antitumor immunity. Several efforts are made to overcome this hostile TME with the development of...

Ubiquitin-proteasome system reveals clinical subtypes with distinct molecular characteristics and prognoses in gastric cancer

Modulators in ubiquitin-proteasome system (UPS) has been implicated in regulating cancer-related genes, immune responses, and oncogenesis. However, the global UPS expression pattern and its role in gastric cancer (GC) pathology remain elusive. Herein, we integrated the modulators in UPS and dissected their associations with tumor microenvironment (TME), therapeutic response and prognosis in GC. Ten eligible GC cohorts (n = 2161) were collected in this comprehensive analysis. Unsupervised clustering based on expression profile of ubiquitination regulators was performed to identify distinct expression pattern. Then, pathway activation, and TME characteristics and prognosis were explored for patients in each pattern. Finally, a UPS scoring system in GC, termed UPSGC, is developed for individualized quantification of UPS expression pattern. Two prognosis-distinctive UPS expression patterns were identified and validated. Multiple interdependent characteristics were found in each pattern. Patients in the pattern with poor prognosis were found with activation of EMT, TNFα/NF-κB and IL6/JAK/STAT3 signaling, and more infiltration of immunosuppressive M2 macrophages and Th2 cells in TME. And another pattern was characterized by upregulation of angiogenesis, Notch and Wnt-β/catenin signaling, as well as enrichment of microvessels in TME. Based on the UPSGC system, two pattern-related clinical subtypes were identified. Finally, the UPSGC subtypes were validated as robust biomarker to predict patient's therapeutic responses and survival outcomes. In conclusion, this study proposes two previously unexplored UPS expression patterns in GC, in which patients have distinct survival outcomes and molecular characteristics. The findings provide new evidences to support the clinical relevance of ubiquitination with personalized therapy.

Toll-like Receptor-Mediated Immunomodulation of Th1-Type Response Stimulated by Recombinant Antigen of Type 2 Porcine Reproductive and Respiratory Syndrome Virus (PRRSV-2).

PRRSV infects CD163-positive macrophages and skews their polarization toward an M2 phenotype, followed by T-cell inactivation. In our previous study, we found that recombinant protein A1 antigen derived from PRRSV-2 was a potential vaccine or adjuvant for immunization against PRRSV-2 infection due to its ability to repolarize macrophages into M1 subtype, thereby reducing CD163 expression for viral entry and promoting immunomodulation for Th1-type responses, except for stimulating Toll-like receptor (TLR) activation. The aim of our current study was to evaluate the effects of another two recombinant antigens, A3 (ORF6L5) and A4 (NLNsp10L11), for their ability to trigger innate immune responses including TLR activation. We isolated pulmonary alveolar macrophages (PAMs) from 8- to 12-week-old specific pathogen free (SPF) piglets and stimulated them with PRRSV (0.01 MOI and 0.05 MOI) or antigens. We also investigated the T-cell differentiation by immunological synapse activation of PAMs and CD4+ T-cells in the cocultured system. To confirm the infection of PRRSV in PAMs, we checked the expression of TLR3, 7, 8, and 9. Our results showed that the expression of TLR3, 7, and 9 were significantly upregulated in PAMs by A3 antigen induction, similar to the extent of PRRSV infection. Gene profile results showed that A3 repolarizes macrophages into the M1 subtype potently, in parallel with A1, as indicated by significant upregulation of proinflammatory genes (TNF-α, IL-6, IL-1β and IL-12). Upon immunological synapse activation, A3 potentially differentiated CD4 T cells into Th1 cells, determined by the expression of IL-12 and IFN-γ secretion. On the contrary, antigen A4 promoted regulatory T cell (T-reg) differentiation by significant upregulation of IL-10 expression. Finally, we concluded that the PRRSV-2 recombinant protein A3 provided better protection against PRRSV infection, suggested by its capability to reeducate immunosuppressive M2 macrophages into proinflammatory M1 cells. As M1 macrophages are prone to be functional antigen-presenting cells (APCs), they can call for TLR activation and Th1-type immune response within the immunological synapse.

Abstract PD1-06: Black patients with triple negative breast cancer (TNBC) have enriched stromal tumor infiltrating lymphocytes (sTILs) and receive preferential benefit from neoadjuvant immunotherapy

Abstract Introduction TNBC is overrepresented in Black women, and Black patients with TNBC have worse clinical outcomes compared to non-Black patients. This disparity likely results from racial differences in clinical, biological, and demographic features of TNBC and social determinants of health. Neoadjuvant chemoimmunotherapy is current standard of care for high-risk TNBC. However, Black patients have been poorly represented in immunotherapy TNBC trials, making it difficult to assess comparative efficacy of immunotherapy in Black patients. Methods We utilized two TNBC neoadjuvant trials to assess racial differences in the tumor immune microenvironment composition and evaluate impact of race on response to chemotherapy vs chemoimmunotherapy. NeoSTOP trial (NCT02413320) randomized 100 stage I-III TNBC patients to receive neoadjuvant carboplatin/paclitaxel + doxorubicin/cyclophosphamide (CbP+AC) or carboplatin/docetaxel (CbD). NeoPACT trial (NCT03639948) enrolled 120 patients with stage I-III TNBC who received neoadjuvant CbD + pembrolizumab (CbD+P). sTILs were centrally quantified, and RNA extracted from pretreatment tissue was subjected to next-generation sequencing. Relative leukocyte fractions were computed by CIBERSORTx. Factors were tested as predictors of pathologic complete response (pCR) using logistic regression analysis. Event-free survival (EFS) was estimated by the Kaplan-Meier method and compared between groups by log-rank test, followed by Cox regression analysis. Results The study population includes 197 patients with known race, sTILs, and gene expression data (84 patients from NeoSTOP, 113 from NeoPACT). 15/84 (18%) patients in NeoSTOP and 20/113 (18%) patients in NeoPACT self-reported Black race. There was no significant difference in age, T or N stage, or germline BRCA1/2 mutation status by race in either study. Black patients had significantly higher sTILs than non-Black patients (median 40% vs 15%, P=0.048) and were more likely to have ≥20% sTILs than non-Black patients (66% vs 44%, P=0.026). There was no significant difference in pCR by race in NeoSTOP (OR=0.60, 95% CI 0.19-1.84, P=0.37; pCR 47% for Black vs 59% for non-Black). In contrast, in NeoPACT, Black patients had a significantly higher pCR compared to non-Black patients (OR=3.27, 95% CI 1.01-10.64, P=0.049; pCR 79% for Black vs 53% for non-Black). In NeoSTOP, EFS was similar for Black and non-Black patients (3-year EFS 92% and 94%, respectively, HR=0.88, 95% CI 0.11-7.28, P=0.90). In NeoPACT, EFS was numerically higher in Black vs non-Black patients (3-year EFS 93% and 81%, respectively, HR=0.43, 95% CI 0.05-3.36, P=0.40); NeoPACT survival follow-up is ongoing at the time of this report. On CIBERSORTx analysis, Black patients had relative depletion of immunosuppressive pro-tumorigenic M2 macrophages (P=0.005) and CD4+ memory resting T cells (P=0.021) compared to non-Black patients. Conclusions Compared to non-Black patients, Black patients with TNBC are more likely to have immune-enriched tumors with lower relative abundance of immunosuppressive leukocytes. These findings suggest potential for higher relative magnitude of benefit from checkpoint inhibitor therapy in Black compared to non-Black patients. Supporting this biological hypothesis, we noted that Black and non-Black patients had equivalent rates of pCR with neoadjuvant chemotherapy; however, pCR rate among Black patients was significantly higher than in non-Black patients when treated with neoadjuvant chemoimmunotherapy. These findings should be confirmed in other studies and can optimize utilization of neoadjuvant chemoimmunotherapy. Our findings also underscore the importance of efforts to address disparity in access and use of immunotherapy in Black patients. Citation Format: Shane R. Stecklein, Rachel Yoder, Roberto Salgado, Joshua M. Staley, Anne O’Dea, Lauren Nye, Manana Elia, Deepti Satelli, Gregory Crane, Richard McKittrick, Andrew K. Godwin, Qamar Khan, Priyanka Sharma. Black patients with triple negative breast cancer (TNBC) have enriched stromal tumor infiltrating lymphocytes (sTILs) and receive preferential benefit from neoadjuvant immunotherapy [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr PD1-06.

Abstract B040: Leader cells mediate tumor engraftment and promote immunosuppression to drive ovarian cancer progression in vivo

Abstract Epithelial ovarian cancer (EOC) is most frequently diagnosed at an advanced stage, and is characterized by its high recurrence rate and almost-universal acquisition of chemoresistance. Metastasis of EOC is driven by heterogenous spheroids that passively disseminate throughout the peritoneal cavity, promoting tumor spread and recurrence. We previously identified a sub-population of highly motile and invasive “Leader cells” (LCs) that express the basal epithelial protein KRT14 as an absolute determinant of invasive potential. We demonstrated KRT14 knockout in EOC cells does not impact viability or proliferation, however completely ablates their ability to invade through Matrigel in 2D and 3D format in vitro. Although a role for LCs in mesothelial clearance and invasion has been established, these mechanisms remain poorly understood. Here we show a critical role for KRT14+ LCs in immunosuppression and tumor-immune privilege in vivo. In the ID8 syngeneic EOC mouse model, tumor-specific LC (KRT14) loss (LCKO) impaired ovarian intrabursal primary tumor engraftment and subsequent metastatic spread. Immune phenotyping by flow cytometry revealed that mice bearing LCKO tumors displayed decreased proportions of immunosuppressive tumor-adjacent Tregs and M2 macrophages, and improved CD8+ T-cell/Treg ratios. Conversely, forced LC-overexpression (LCOE) in EOC tumors accelerated metastasis, and promoted tumor-immune evasion via upregulated secretion of suppressive chemokines including CCL22 and CCL5. In addition to their critical role in migration and invasion, the current study provides in vivo evidence that leader cells are mediators of immune suppression in ovarian cancer. This work supports the basis that defining novel strategies to target LCs to promote anti-tumor immunity and limit metastatic progression may be a useful strategy for improved epithelial ovarian cancer therapy. Citation Format: Amy L. Wilson, Brittany R. Doran, Bashirah Basri, Laura R. Moffitt, Magdalena Plebanski, Andrew N. Stephens, Maree Bilandzic. Leader cells mediate tumor engraftment and promote immunosuppression to drive ovarian cancer progression in vivo [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr B040.

Characterization of the gene signature correlated with favorable response to chemoradiotherapy in rectal cancer: A hypothesis-generating study.

This study aimed to define the gene signature associated with response to neoadjuvant chemoradiotherapy (nCRT), or chemoradiosensitivity (CRS) signature, in rectal cancer, and investigate the correlation between the CRS signature and characteristics of tumor. Three public microarray datasets of pre-nCRT rectal cancer were used to discover and validate the CRS signature, and the pathway analysis of the CRS signature was performed. Patients in The Cancer Genome Atlas (TCGA) dataset were stratified according to the CRS signature enrichment score, and mutational profile and proportions of infiltrated immune cells were compared. In the discovery dataset (GSE53781), 95 genes were upregulated in complete responders compared to non-complete responders and defined as the CRS signature. Pathways regarding DNA replication and repair processes as well as inflammatory response were enriched in the CRS signature. In the validation datasets (GSE35452 and GSE45404), patients with favorable response to nCRT exhibited higher enrichment score of the CRS. In TCGA-READ cohort, patients with high CRS signature harbored KRAS mutation in lower frequency than those with low CRS signature. In addition, proportions of proinflammatory immune cells were higher, but proportion of immunosuppressive M2 macrophages was lower in patients with high CRS signature than those with low CRS signature. The current integrative bioinformatic analysis suggests the CRS signature and showed that the CRS signature is associated with dissimilar mutational profile and increased immune response. The discovered CRS signature and related characteristics may serve as candidate of stratification factor in upcoming studies for rectal cancer.

Current State of Immunotherapy and Mechanisms of Immune Evasion in Ewing Sarcoma and Osteosarcoma

We argue here that in many ways, Ewing sarcoma (EwS) is a unique tumor entity and yet, it shares many commonalities with other immunologically cold solid malignancies. From the historical perspective, EwS, osteosarcoma (OS) and other bone and soft-tissue sarcomas were the first types of tumors treated with the immunotherapy approach: more than 100 years ago American surgeon William B. Coley injected his patients with a mixture of heat-inactivated bacteria, achieving survival rates apparently higher than with surgery alone. In contrast to OS which exhibits recurrent somatic copy-number alterations, EwS possesses one of the lowest mutation rates among cancers, being driven by a single oncogenic fusion protein, most frequently EWS-FLI1. In spite these differences, both EwS and OS are allied with immune tolerance and low immunogenicity. We discuss here the potential mechanisms of immune escape in these tumors, including low representation of tumor-specific antigens, low expression levels of MHC-I antigen-presenting molecules, accumulation of immunosuppressive M2 macrophages and myeloid proinflammatory cells, and release of extracellular vesicles (EVs) which are capable of reprogramming host cells in the tumor microenvironment and systemic circulation. We also discuss the vulnerabilities of EwS and OS and potential novel strategies for their targeting.

Enhanced lipid metabolism confers the immunosuppressive tumor microenvironment in CD5-positive non-MYC/BCL2 double expressor lymphoma.

Lymphoma cells expressing CD5 (CD5+) confer inferior outcome of diffuse large B-cell lymphoma (DLBCL), especially in non–MYC/BCL2 double expressor (non-DE) patients. In tumor microenvironment, CD5+ non-DE tumor revealed increased proportion of immunosuppressive M2 macrophages and enhanced pathways related to macrophage activation and migration. In accordance to M2 activation, lipid metabolism was upregulated, including fatty acid uptake and fatty acid oxidation, which supplied energy for M2 macrophage polarization and activation. Meanwhile, CD36 expression was upregulated and strongly correlated to the proportion of M2 macrophages in CD5+ non-DE DLBCL. In vitro, a DLBCL cell line (LY10) overexpressing CD5 significantly increased M2 proportion in comparison with control when cocultured with peripheral blood mononuclear cells (PBMCs). The addition of metformin significantly decreased the M2 proportion and the CD36 expression level in the coculture systems, indicating that metformin could target altered lipid metabolism and decrease M2 macrophages in DLBCL, especially in CD5+ non-DE lymphoma. In conclusion, enhanced lipid metabolism and M2 macrophage activation contributed to the immunosuppressive tumor microenvironment and could be potential therapeutic targets in CD5+ non-DE DLBCL.

2D Copper(II) Metalated Metal-Organic Framework Nanocomplexes for Dual-enhanced Photodynamic Therapy and Amplified Antitumor Immunity.

The immunosuppressive tumor microenvironment (TME) poses tremendous challenges for efficient immunotherapy. Smart nanomedicine is designed to modulate immunosuppressive TMEs based on the combination of dual-enhanced photodynamic therapy (PDT) triggered immunogenic cell death (ICD) and relieved hypoxic microenvironment. Copper(II) metalated metal-organic framework nanosheets (Cu-TCPP(Al)) are the foundation of the nanomedicine, and platinum nanoparticles (Pt NPs) and folate are subsequently introduced onto the Cu-TCPP(Al) surface (Cu-TCPP(Al)-Pt-FA). Upon targeted cellular uptake, intracellular GSH concentration is decreased because of the specific adsorption between GSH and CuII; meanwhile, Pt NPs possess catalase-like activity, which can continuously depose intracellular H2O2 to O2 to alleviate the hypoxic TME. The two factors synergistically improve the ROS concentration for dual-enhanced PDT. The highly toxic ROS can correspondingly cause amplified oxidative stress and then trigger the ICD. The ICD process stimulates antigen-presenting cells and activates the systemic antitumor immune response. Furthermore, the relieved hypoxic TME increases the infiltration of cytotoxic T lymphocytes (CTLs) at the tumor site, which can promote the transformation of the immunosuppressive M2 macrophage to immunoactive M1 phenotype. The easily prepared yet versatile nanomedicine possesses an excellent antitumor effect with the cooperation of dual-enhanced PDT and immunotherapy.

Oligo-Fucoidan supplementation enhances the effect of Olaparib on preventing metastasis and recurrence of triple-negative breast cancer in mice

BackgroundSeaweed polysaccharides have been recommended as anticancer supplements and for boosting human health; however, their benefits in the treatment of triple-negative breast cancers (TNBCs) and improving immune surveillance remain unclear. Olaparib is a first-in-class poly (ADP-ribose) polymerase inhibitor. Oligo-Fucoidan, a low-molecular-weight sulfated polysaccharide purified from brown seaweed (Laminaria japonica), exhibits significant bioactivities that may aid in disease management.MethodsMacrophage polarity, clonogenic assays, cancer stemness properties, cancer cell trajectory, glucose metabolism, the TNBC 4T1 cells and a 4T1 syngeneic mouse model were used to inspect the therapeutic effects of olaparib and Oligo-Fucoidan supplementation on TNBC aggressiveness and microenvironment.ResultsOlaparib treatment increased sub-G1 cell death and G2/M arrest in TNBC cells, and these effects were enhanced when Oligo-Fucoidan was added to treat the TNBC cells. The levels of Rad51 and programmed death-ligand 1 (PD-L1) and the activation of epidermal growth factor receptor (EGFR) and adenosine 5′-monophosphate (AMP)-activated protein kinase (AMPK) facilitate drug resistance and TNBC metastasis. However, the combination of olaparib and Oligo-Fucoidan synergistically reduced Rad51 and PD-L1 levels, as well as the activity of EGFR and AMPK; consistently, TNBC cytotoxicity and stemness were inhibited. Oligo-Fucoidan plus olaparib better inhibited the formation of TNBC stem cell mammospheroids with decreased subpopulations of CD44high/CD24low and EpCAMhigh cells than monotherapy. Importantly, Oligo-Fucoidan plus olaparib repressed the oncogenic interleukin-6 (IL-6)/p-EGFR/PD-L1 pathway, glucose uptake and lactate production. Oligo-Fucoidan induced immunoactive and antitumoral M1 macrophages and attenuated the side effects of olaparib, such as the promotion on immunosuppressive and protumoral M2 macrophages. Furthermore, olaparib plus Oligo-Fucoidan dramatically suppressed M2 macrophage invasiveness and repolarized M2 to the M0-like (F4/80high) and M1-like (CD80high and CD86high) phenotypes. In addition, olaparib- and Oligo-Fucoidan-pretreated TNBC cells resulted in the polarization of M0 macrophages into CD80(+) M1 but not CD163(+) M2 macrophages. Importantly, olaparib supplemented with oral administration of Oligo-Fucoidan in mice inhibited postsurgical TNBC recurrence and metastasis with increased cytotoxic T cells in the lymphatic system and decreased regulatory T cells and M2 macrophages in tumors.ConclusionOlaparib supplemented with natural compound Oligo-Fucoidan is a novel therapeutic strategy for reprogramming cancer stemness, metabolism and the microenvironment to prevent local postsurgical recurrence and distant metastasis. The combination therapy may advance therapeutic efficacy that prevent metastasis, chemoresistance and mortality in TNBC patients.

Biomimetic O2 Self-generated hybrid membrane nanoplatform for blocking the polarization towards immunosuppressive M2 macrophage phenotype and enhancing sonodynamics therapy in orthotopic colorectal cancer

Colorectal cancer is one of the most common intestinal tumors, of which occurrence and development are closely related to immune escape activity in vivo. Then, how to comprehensively block immune escape remains a big challenge for immunotherapy. Thus, a biomimetic nanoprobe Ce6@ZIF-8/hybrid membrane/PA (CZ@HP) is designed by coating Ce6 loaded ZIF-8 with a hybrid membrane composed of neutrophil membrane (Nm) & red blood cell membrane (Rm), accompanied by palmitoyl ascorbate (PA) to achieve reprogramming of immunosuppressive tumor microenvironment (TME) and enhancement of sonodynamics therapy (SDT) in hypoxic tumor. Cell and animal experiments indicate that, the CZ@HP probe can specifically target tumor at the inflammatory site, and it can effectively reduce the adsorption of neutrophils, then inhibit the polarization toward immunosuppressive M2 macrophage phenotype. Immunofluorescence staining and ELISA experiments demonstrate that PA and Rm can generate O2 and significantly improve the hypoxic environment, it enhances the polarization of anti-tumor M1 macrophages and regulate related cytokines, meanwhile improves the therapeutic effect of SDT. The survival rate of mice is elevated to 100% within 60 d. Overall, this O2 self-generated biomimetic hybrid membrane nanoprobe can block immune escape from various aspects and obtain enhanced SDT effect, then increase the treatment efficiency of inflammatory tumor, significantly improve the prognosis and elevate the survival rate of mice, providing a new idea for the clinical diagnosis and treatment of colitis-associated tumors.

Mutant p53 drives an immune cold tumor immune microenvironment in oral squamous cell carcinoma

The critical role of the tumor immune microenvironment (TIME) in determining response to immune checkpoint inhibitor (ICI) therapy underscores the importance of understanding cancer cell–intrinsic mechanisms driving immune-excluded (“cold”) TIMEs. One such cold tumor is oral cavity squamous cell carcinoma (OSCC), a tobacco-associated cancer with mutations in the TP53 gene which responds poorly to ICI therapy. Because altered TP53 function promotes tumor progression and plays a potential role in TIME modulation, here we developed a syngeneic OSCC models with defined Trp53 (p53) mutations and characterized their TIMEs and degree of ICI responsiveness. We observed that a carcinogen-induced p53 mutation promoted a cold TIME enriched with immunosuppressive M2 macrophages highly resistant to ICI therapy. p53-mutated cold tumors failed to respond to combination ICI treatment; however, the combination of a programmed cell death protein 1 (PD-1) inhibitor and stimulator of interferon genes (STING) agonist restored responsiveness. These syngeneic OSCC models can be used to gain insights into tumor cell–intrinsic drivers of immune resistance and to develop effective immunotherapeutic approaches for OSCC and other ICI-resistant solid tumors.

The human cathelicidin peptide LL-37 inhibits pancreatic cancer growth by suppressing autophagy and reprogramming of the tumor immune microenvironment

Pancreatic cancer is amongst the most lethal malignancies, while its poor prognosis could be associated with promotion of autophagy and the tumor immune microenvironment. Studies have confirmed the pro-tumorigenic nature of the cathelicidin family of peptide LL-37 in several types of cancer. However, at higher doses, LL-37 exerts significant cytotoxicity against gastrointestinal cancer cells. In our study, we investigated the anti-tumorigenic potential of LL-37 in pancreatic cancer and the underlying mechanisms. Our results have shown that LL-37 inhibited the growth of pancreatic cancer both in vitro and in vivo. Mechanistic studies have demonstrated that LL-37 induced DNA damage and cell cycle arrest through induction of reactive oxygen species (ROS). Further study indicates that LL-37 suppressed autophagy in pancreatic cancer cells through activation of mTOR signaling, leading to more accumulation of ROS production and induction of mitochondrial dysfunctions. With combined treatment of LL-37 with the mTOR inhibitor rapamycin, LL-37-induced ROS production and cancer cell growth inhibition were attenuated. Subsequent in vivo study has shown that LL-37 downregulated the immunosuppressive myeloid-derived suppressor cells and M2 macrophages while upregulated the anti-cancer effectors CD8+ and CD4+ T cells in the tumor microenvironment. By using an in vitro co-culture system, it was shown that promotion of M2 macrophage polarization would be suppressed by LL-37 with inhibition of autophagy, which possessed significant negative impact on cancer growth. Taken together, our findings implicate that LL-37 could attenuate the development of pancreatic cancer by suppressing autophagy and reprogramming of the tumor immune microenvironment.

PERK promotes immunosuppressive M2 macrophage phenotype by metabolic reprogramming and epigenetic modifications through the PERK-ATF4-PSAT1 axis.

The endoplasmic reticulum (ER) is a specialized organelle that participates in multiple cellular functions including protein folding, maturation, trafficking, and degradation to maintain homeostasis. However, hostile conditions in the tumor microenvironment (TME) disturb ER homeostasis. To overcome these conditions, cells activate ER stress response pathways, which are shown to augment the suppressive phenotypes of immune cells; however, the molecular mechanisms underpinning this process remain elusive. Here, we discuss a recent study by Raines et al, that suggests the role of the helper T-cell 2 (TH2) cytokine interleukin-4 (IL-4), and the TME in facilitating a protein kinase RNA-like ER kinase (PERK)-signaling cascade in macrophages, which promotes immunosuppressive M2 macrophage activation and proliferation. Further, the authors showed that PERK signaling promotes both mitochondrial respirations to fulfill cellular energy requirements and signaling through ATF4, which regulate phosphoserine aminotransferase 1 (PSAT1) activity to mediate the serine biosynthesis pathway. These results highlight a previously uncharacterized role for PERK in cellular metabolism and epigenetic modification in M2 macrophages, and thus offers a new therapeutic strategy for overcoming the immunosuppressive effects in the TME.

Babesia microti Infection Inhibits Melanoma Growth by Activating Macrophages in Mice.

Babesia microti is an obligate intraerythrocytic protozoan transmitted by an Ixodes tick. Infections caused by protozoa, including Plasmodium yoelii and Toxoplasma gondii, are shown to inhibit tumor development by activating immune responses. Th1 immune response and macrophages not only are essential key factors in Babesia infection control but also play an important role in regulating tumor development. In this study, we investigated the effects of B. microti infection on melanoma in tumor-bearing mice. The results showed that B. microti infection could inhibit the growth of melanoma, significantly enlarge the spleen size (p ≤ 0.0001), and increase the survival period (over 7 days) of tumor-bearing mice. Mouse spleen immune cell analysis revealed that B. microti-infected tumor-bearing mice could increase the number of macrophages and CD4+ T cells, as well as the proportion of CD4+ T cells and M1 macrophages in the tumor. Immunohistochemical assays showed that B. microti infection could inhibit tumor angiogenesis (p ≤ 0.0032). Meanwhile, both B. microti-infected erythrocytes and culture supernatant were observed to significantly (p ≤ 0.0021) induce the mRNA expression of iNOS, IL-6, and TNF-α in macrophages. Moreover, B. microti culture supernatant could also repolarize IL-4-induced M2 macrophages to the M1 type. Overall, B. microti exerted antitumor effects by stimulating the immune system of tumor-bearing mice and inducing the polarization of immunosuppressive M2 macrophages to pro-inflammatory M1 macrophages.

Abstract 4155: Mitazalimab, a potent CD40 agonist in combination with chemotherapy redirects and activates tumor infiltrating myeloid cells

Abstract Mitazalimab is a human CD40 agonistic antibody developed for immunotherapy of cancer. Activation of CD40, expressed on myeloid cells, such as dendritic cells and tumor infiltrating macrophages, leads to improved T cell priming and initiation of T cell-dependent anti-tumor responses. Mitazalimab is a FcγR crosslinking-dependent IgG1 antibody, with potential for high efficacy and manageable safety profile. Immunologically cold and immune-excluded tumors, such as pancreatic cancer, are defined by low infiltration of immune cells as well as low expression and release of neoantigens. In pancreatic cancer, effector CD8+ T cells are excluded from the tumor by the desmoplastic tumor stroma, which surrounds the tumor and hosts immunosuppressive macrophages that dampen the immune response in the tumor microenvironment. By activating and re-directing tumor infiltrating myeloid cells, CD40 agonists such as mitazalimab, have the potential to augment the response to chemotherapy and spark an effective anti-tumor response by i) priming T cells reactive to the tumor neoantigens released by the chemotherapy, and ii) inducing degradation of the stroma surrounding the tumor thereby enhancing the efficacy of chemotherapy. The ability of mitazalimab to augment the response to chemotherapy was demonstrated in mice, transgenic for human CD40 (hCD40tg), inoculated with the syngeneic tumor cell line MB49. Mitazalimab, administered repeatedly together with FOLFIRINOX (oxaliplatin, irinotecan, 5-fluorouracil and folinic acid), synergized effectively with chemotherapy, inducing long-term survival. The combined treatment improved activation of antigen presenting cells in the circulation, intratumoral T cell responses as well as reduced the amount of intratumoral immunosuppressive M2 macrophages. By converting the MB49 tumor cell line resistant to FOLFIRINOX treatment, we could further demonstrate that the combination of mitazalimab and FOLFIRINOX induced a strong anti-tumor activity also in a chemoresistant variant of the MB49 cell line. In conclusion, mitazalimab synergizes effectively with chemotherapy, leading to induction of long-term survival in a preclinical tumor model by reducing immunosuppressive M2 macrophages and improving T cell responses intratumorally. These preclinical data, together with the clinical data of mitazalimab from the phase 1 study (NCT02829099), where mitazalimab was well tolerated up to 1200 μg/kg with a manageable safety profile, support the ongoing clinical phase 2 study OPTIMIZE-1 (NCT04888312) of mitazalimab in combination with chemotherapy in pancreatic cancer. Citation Format: Karin Enell Smith, Mia Thagesson, Anneli Nilsson, Doreen Werchau, Peter Ellmark. Mitazalimab, a potent CD40 agonist in combination with chemotherapy redirects and activates tumor infiltrating myeloid cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4155.

Collagen Family and Other Matrix Remodeling Proteins Identified by Bioinformatics Analysis as Hub Genes Involved in Gastric Cancer Progression and Prognosis

Gastric cancer has remained in the top five cancers for over ten years, both in terms of incidence and mortality due to the shortage of biomarkers for disease follow-up and effective therapies. Aiming to fill this gap, we performed a bioinformatics assessment on our data and two additional GEO microarray profiles, followed by a deep analysis of the 40 differentially expressed genes identified. PPI network analysis and MCODE plug-in pointed out nine upregulated hub genes coding for proteins from the collagen family (COL12A1, COL5A2, and COL10A1) or involved in the assembly (BGN) or degradation of collagens (CTHRC1), and also associated with cell adhesion (THBS2 and SPP1) and extracellular matrix degradation (FAP, SULF1). Those genes were highly upregulated at the mRNA and protein level, the increase being correlated with pathological T stages. The high expression of BGN (p = 8 × 10−12), THBS2 (p = 1.2 × 10−6), CTHRC1 (p = 1.1 × 10−4), SULF1 (p = 3.8 × 10−4), COL5A1 (p = 1.3 × 10−4), COL10A1 (p = 5.7 × 10−4), COL12A1 (p = 2 × 10−3) correlated with poor overall survival and an immune infiltrate based especially on immunosuppressive M2 macrophages (p-value range 4.82 × 10−7–1.63 × 10−13). Our results emphasize that these genes could be candidate biomarkers for GC progression and prognosis and new therapeutic targets.

반하 추출물의 종양연관대식세포 조절을 통한 암세포 이동능 저해 효과

The tuber of Pinellia ternata (Thunb.) Brei (TPT) used in traditional Oriental medicine for the treatment of cough, sputum, vomiting, and insomnia, possesses antioxidant, antibacterial, and anti-inflammatory effects. Although recent studies have reported the anticancer effects of TPT in several cancer cells, it is still unclear whether TPT regulates tumor-associated macrophage (TAM) characterized by the immunosuppressive M2 macrophage phenotype. Our results showed that the ethanol extract of TPT (ETPT) suppressed the migration of RAW264.7 mouse macrophage cells and THP-1 human monocytes differentiated into macrophages towards the conditioned media (CM) collected from lung cancer cells, suggesting that ETPT would attenuate the recruitment of macrophages into tumors. In addition, ETPT suppressed the interleukin (IL)-4 or IL-6-induced M2 macrophage polarization in RAW264.7 cells. ETPT treatment not only downregulated the mRNA expression of M2 macrophage markers including arginase-1, mannose receptor C type 1 (MRC-1), and IL-10, but also inhibited the phosphorylation of signal transducer and activator of transcription 3 (STAT3) and STAT6, general regulators of M2 macrophage polarization. Finally, the transwell assay results showed that the CM from M2-polarized RAW264.7 cells increased the migration of mouse lewis lung carcinoma (LLC) cells, while those from RAW264.7 cells co-treated with ETPT and IL-6 significantly reduced the migration of LLC cells. Taken together, our observations clearly demonstrate that ETPT suppressed the cancer cell migration by regulating macrophage recruitment and M2 macrophage polarization.

Stromal HIF2 Regulates Immune Suppression in the Pancreatic Cancer Microenvironment

BACKGROUND & AIMS:Pancreatic ductal adenocarcinoma (PDAC) has a hypoxic, immunosuppressive stroma that contributes to its resistance to immune checkpoint blockade therapies. The hypoxia-inducible factors (HIFs) mediate the cellular response to hypoxia, but their role within the PDAC tumor microenvironment remains unknown.METHODS:We used a dual recombinase mouse model to delete Hif1α or Hif2α in α-smooth muscle actin–expressing cancer-associated fibroblasts (CAFs) arising within spontaneous pancreatic tumors. The effects of CAF HIF2α expression on tumor progression and composition of the tumor microenvironment were evaluated by Kaplan-Meier analysis, reverse transcription quantitative real-time polymerase chain reaction, histology, immunostaining, and by both bulk and single-cell RNA sequencing. CAF-macrophage crosstalk was modeled ex vivo using conditioned media from CAFs after treatment with hypoxia and PT2399, an HIF2 inhibitor currently in clinical trials. Syngeneic flank and orthotopic PDAC models were used to assess whether HIF2 inhibition improves response to immune checkpoint blockade.RESULTS:CAF-specific deletion of Hif2α, but not Hif1α, suppressed PDAC tumor progression and growth, and improved survival of mice by 50% (n ¼ 21–23 mice/group, Log-rank P ¼ .0009). Deletion of CAF-HIF2 modestly reduced tumor fibrosis and significantly decreased the intratumoral recruitment of immunosuppressive M2 macrophages and regulatory T cells. Treatment with the clinical HIF2 inhibitor PT2399 significantly reduced in vitro macrophage chemotaxis and M2 polarization, and improved tumor responses to immunotherapy in both syngeneic PDAC mouse models.CONCLUSIONS:Together, these data suggest that stromal HIF2 is an essential component of PDAC pathobiology and is a druggable therapeutic target that could relieve tumor microenvironment immunosuppression and enhance immune responses in this disease.