Immune-suppressive Macrophages Research Articles

Antibodies Targeting Human or Mouse VSIG4 Repolarize Tumor-Associated Macrophages Providing the Potential of Potent and Specific Clinical Anti-Tumor Response Induced across Multiple Cancer Types.

V-set immunoglobulin domain-containing 4 (VSIG4) is a B7 family protein with known roles as a C3 fragment complement receptor involved in pathogen clearance and a negative regulator of T cell activation by an undetermined mechanism. VSIG4 expression is specific for tumor-associated and select tissue-resident macrophages. Increased expression of VSIG4 has been associated with worse survival in multiple cancer indications. Based upon computational analysis of transcript data across thousands of tumor and normal tissue samples, we hypothesized that VSIG4 has an important role in promoting M2-like immune suppressive macrophages and that targeting VSIG4 could relieve VSIG4-mediated macrophage suppression by repolarizing tumor-associated macrophages (TAMs) to an inflammatory phenotype. We have also observed a cancer-specific pattern of VSIG4 isoform distribution, implying a change in the functional regulation in cancer. Through a series of in vitro, in vivo, and ex vivo assays we demonstrate that anti-VSIG4 antibodies repolarize M2 macrophages and induce an immune response culminating in T cell activation. Anti-VSIG4 antibodies induce pro-inflammatory cytokines in M-CSF plus IL-10-driven human monocyte-derived M2c macrophages. Across patient-derived tumor samples from multiple tumor types, anti-VSIG4 treatment resulted in the upregulation of cytokines associated with TAM repolarization and T cell activation and chemokines involved in immune cell recruitment. VSIG4 blockade is also efficacious in a syngeneic mouse model as monotherapy as it enhances efficacy in combination with anti-PD-1, and the effect is dependent on the systemic availability of CD8+ T cells. Thus, VSIG4 represents a promising new target capable of triggering an anti-cancer response via multiple key immune mechanisms.

Immune infiltrate characterization and differential gene expression by RNA-sequencing analysis in patients with oncogene-addicted NSCLC with early progression under targeted therapy.

8535 Background: Despite remarkable advances with tyrosine kinase inhibitors (TKI), patients (pts) with oncogene addicted advanced non-small cell lung cancer (aNSCLC) eventually face resistance. Moreover, some pts exhibit early TKI resistance, often linked to a reduced response subsequent TKIs. These pts might have unique biology, and identifying post-resistance treatments for them is an unmet need. Here, we explore the transcriptomic profile of pts with aNSCLC and early TKI progression, as compared to long responders. Methods: We included pts with aNSCLC harboring EGFR mutations or oncogenic fusions ( ALK, ROS1, RET, NTRK), treated with TKI at Gustave Roussy and enrolled in the prospective MATCH R trial (NCT02517892). Clinical data on treatment outcomes and survival were collected. Pts with PFS < 6 months were considered as having early progression (EP), pts with PFS > 18 months as long responders (LR). At least one tissue biopsy was performed at PD, on which bulk RNA sequencing was carried out. We explored the transcriptomic differences between pts with EP and LR. Differential gene expression (DESeq2), pathway enrichment studies and identification with MSigDb were performed and compared between groups. Immune cell type contents were evaluated by CIBERSORT deconvolution method. Results: From 2015 to 2022, 37 pts were included. 68% were females, median age was 55 (IQR43-83). 18 carried EGFR mutations (EP N=5, 28%; LR N=13, 72%), 19 had fusions (EP N=10, 52%; LR N=9, 48%). All pts had progressed to targeted TKI (7 first generation EGFR-TKI, 11 osimertinib, 8 crizotinib, 7 second generation ALK-TKI, 2 lorlatinib, 2 pralsetinib, 1 repotrectinib). Immune suppressive macrophages were the most represented immune cells in EGFR+ and fusion+ PD samples (33.5% and 33.2% of immune cells). The composition of the immune infiltrate was not significantly different between EP and LR in EGFR+ and fusion+ pts. DESeq2 showed that EGFR+ EP had significantly lower EPCAM(log2FC: 1.60, padj=0.01), higher SOS1 (log2FC: -1.18, padj=0.01), higher JAK2(log2FC: -1.56, padj=0.02) expression, as compared to EGFR+ LR. In the fusion+ group, EP had higher FGFR3/4 (log2FC: -3.5, padj=0.02) and lower MTAP expression (log2FC: 1.75, padj=0.03). Hallmark analysis in the EGFR+ showed higher activation of TNFα, IFNγ response, and KRAS pathways in EP (NES: -1.72, p=0.03; NES=-1.78,p=0.02; NES:-2.07, p=0.01) versus LR. No differential activation was observed in the fusion+ group. Conclusions: Immune suppressive macrophages are the most represented immune cells at PD to TKI, irrespective of the treatment duration. Pts with EP seem to have a different biology, with transcriptomic features of stemness and immune suppression in the EGFR+ pts, and lower MTAP expression in the fusion+ group. This should be further explored for the development of novel therapeutics.

Abstract PO5-05-05: Clinical outcomes of CDK4/6 inhibitors in patients with bone only metastatic breast cancer

Abstract Background: CDK4/6 inhibitors (CDK 4/6i) have significantly advanced the treatment of hormone receptor-positive (HR+) metastatic breast cancer in the last decade. Several clinical trials including PALOMA, MONALEESA, and MONARCH have shown improved progression-free survival (PFS) rates when CDK4/6i were combined with endocrine therapy (ET). These results led to recommending their use in combination with ET as the first line of therapy in the metastatic settings. Bone metastasis is the most common site of disease in patients with HR+ metastatic breast cancer, about 40% of whom have bone as the only site of metastasis (bone-only disease [BoD]). While previous studies have shown that BoD can have a unique clinical course and a more favorable prognosis compared to non-BoD metastasis, most of the aforementioned CDK 4/6i clinical trials have not shown significant improvements in PFS in BoD subset of patients. Here we aimed to investigate the survival outcomes of BoD patients at a single institution center and evaluate CDK 4/6i activity in preclinical models of bone metastasis in breast cancer. Patients and methods: Patients with HR+, HER2-negative metastatic breast cancer from a retrospective CDK4/6i study at Washington University School of Medicine were included in this analysis. We selected all the patients who received CDK4/6i in the first- or second-line settings from June 2014 to March 2020. Outcomes were collected through June 10, 2023. The Kaplan-Meier method with log rank test and multivariable cox model were used for survival analysis. Results: 114 female patients (n=75 first line, n=39 second line, n=20 African American, n=94 Caucasian) were included in this study. All patients received Palbociclib as the first exposure to CDK4/6 inhibitor in combination with ET for at least two months. Median age was 60 years (range, 28-87) and forty-seven patients (41%) were in the BoD group. Survival outcomes for BoD group were compared to the rest of patients who had either bone and visceral or visceral only metastatic lesions (BV group). Our results demonstrated a significantly reduced median PFS in BoD group compared to BV group (13.1 months [95%CI:8-18.2] vs 19.6 months [95%CI:6.8-32.4], p=0.01) respectively. There was no significant difference in overall survival between BoD and BV groups (36.6 months [95%CI:32.2-41] vs 41.7 months [95%CI 36.1-47.2] p=0.74) respectively. In multivariate analysis regarding the associated risk factors with PFS, after adjusting for age and race, BoD had a HR of 1.62 (95%CI:1.04-2.54) for PFS. Among the 75 patients who received CDK4/6i as their first line of therapy, BoD (n=32 patients [42.7%]) also showed an inferior (p=0.05) median PFS (20 months [95%CI 12.9-27]) compared to BV group (n=43 patients [57.3%], median PFS=28.4 months [95%CI: 16.3-40.4]). To investigate potential treatment resistance mechanisms in bone metastases, we used the triple negative 4T1 and the ER+ luminal B PyMT Bo1 breast tumor cell line derived mammary gland models in immunocompetent mice. Single agent CDK4/6i, ribociclib, decreased primary mammary gland tumor burden. This effect was abrogated when CD8+ T-cells were depleted. In the bone colonization model using these cell lines, ribociclib had little effect on bone tumor burden. Arg1+ immune suppressive macrophages were significantly decreased in the primary mammary tumor but not in the bone tumor after ribociclib administration, suggesting a relative treatment resistance and immune suppression in the bone tumor microenvironment compared to the primary mammary gland. Conclusion: CDK4/6i play a significant role in the treatment of patients with HR+ metastatic breast cancer. Our single institution, retrospective study and preclinical data suggests that CDK4/6i may not be as effective for the treatment of tumors residing in the bone microenvironment, warranting future studies to understand the mechanisms of CDK4/6 inhibitors action in bone versus visceral metastases. Citation Format: Mahta Mardani, Suleyman Noordeen, Katherine Clifton, Cynthia Ma, Jingqin Luo, Jing Xi, Nusayba Bagegni, Foluso Ademuyiwa, Rama Suresh, Ashley Frith, Andrew Davis, Ron Bose, Lindsay Peterson, Shana Thomas, Yu Tao, Takayuki Kobayashi, Jingyu Xiang, Yalin Xu, Xinming Su, Katherine Weilbaecher. Clinical outcomes of CDK4/6 inhibitors in patients with bone only metastatic breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-05-05.

Abstract 7577: A new cancer therapy strategy: Synthetic lethality induced by inhibition of mTOR and cMYC and JNK activation

Abstract Cancer cells are characterized by aberrant signal transduction and cell cycle control pathways that lead to unchecked cell proliferation. Uncontrolled cancer cell proliferation, as well as immune suppressive macrophage proliferation, contribute to tumor growth and spread. Effort to suppress tumor cell growth using Inhibitors of PI3K or MEK signaling pathways can initially inhibit tumor growth, but resistance develops due to upregulation of the alternative pathway. We identified a novel series of allosteric tyrosine kinase receptor inhibitors that induce synthetic lethality in AML, lung carcinoma, pancreatic carcinoma, and other tumor cells and in proliferating macrophages in vitro and in vivo by simultaneously inhibiting cMyc and Tor and activating the stress activated kinase JNK, leading to p53 mediated cell cycle arrest. Select kinase inhibitors with a unique positioning of a single side chain inhibited CDK8/19 and thereby Myc while stimulating phosphorylation and activation of JNK and p38. JNK activation promoted sustained ATF-2 and cJun-mediated p53 and Chk1/Chk2 activation, thereby promoting cell cycle arrest in G2/M and ultimately, apoptosis. These inhibitors potently suppressed tumor growth in vivo by halting both tumor cell and macrophage proliferation in G2/M. They synergized with anti-PD-1 and anti-CTLA-4 and led to tumor eradication and immunological memory formation. These studies identify novel allosteric inhibitors with potent synthetically lethal anti-tumor activity. Citation Format: Hui Chen, Jason Rivera, Marc Paradise, Zihan Xu, Giuliana Mognol, Judith Varner. A new cancer therapy strategy: Synthetic lethality induced by inhibition of mTOR and cMYC and JNK activation [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 7577.

Abstract 6516: Single-cell protein activity inference reveals lymphocytic immune infiltration in diffuse intrinsic pontine glioma induced by focused ultrasound

Abstract Diffuse intrinsic pontine glioma (DIPG) is a highly aggressive pediatric brain tumor, located in the brainstem, and traditionally thought to be immune-excluded with no significant infiltration by cytotoxic T-cells, and therefore not amenable to immunotherapies. Focused ultrasound technology (FUS) for drug delivery is being investigated for DIPG in both preclinical and clinical trial settings. In this study, we sought to study the effects of FUS and radiation therapy (RT) on immune cell infiltration in DIPG tumors to assess their ability to alter the immune microenvironment and potentially enhance combination immunotherapies. Specifically, we generated single-cell RNAseq profiles for 27,780 cells from syngeneic orthotopic DIPG tumors (KPAP cell line containing H3K27M mutation, ATRX and P53 loss, and PDGFRA overexpression) after 14 days of tumor growth across the following treatment arms: vehicle control, FUS, and RT (n=2 each group). FUS was applied by 4-point sonication around the tumor with a pressure of 0.6 Mpa at days 8 and 11 post tumor implantation (PTI). RT was administered in fractions of 3 Gy daily from days 8 to 11 PTI (12Gy total). We leveraged a set of algorithms for network-based inference of regulatory protein activity to infer a set of context-matched gene regulatory networks from which protein activity can be inferred by expression of downstream targets. By this approach, we identified distinct clusters of immune cells segregating by cell type as inferred by singleR and activity of known lineage markers. Strikingly, we found that both RT and FUS significantly depleted a TGFb-expressing population of immune-suppressive macrophages as compared to vehicle controls. Importantly, FUS only additionally increased T-cell infiltration into the tumor micro-environment over three-fold as compared to vehicle controls, which was not observed in response to RT. Further protein validation is underway. This has significant implications for therapeutic combinations of FUS-mediated drug delivery with immune checkpoint inhibitors in DIPG, such that in addition to drug delivery, FUS-mediated immune modulatory effects may provide more proinflammatory response to enhance immunotherapy. Citation Format: Erin White, Hong-Jian Wei, Ester Calvo Fernández, Hanna Minns, Jovana Pavisic, Robyn Gartrell, Cheng-Chia Wu, Andrea Califano, Aleksandar Obradovic. Single-cell protein activity inference reveals lymphocytic immune infiltration in diffuse intrinsic pontine glioma induced by focused ultrasound [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 6516.

O214: The tumour immune microenvironment and its response to exercise during treatment for Oesophagogastric cancer

Abstract Introduction Pre-clinical studies suggest that exercise during cancer treatment may enhance tumour regression and promote an anti-tumorigenic immune microenvironment. Considering the evolution of immunotherapy in oesophagogastric cancer, further understanding of the host-tumour immune response to exercise therapy is of significant interest. Methods Using multiplex immunofluorescence, resection specimens of 30 patients with oesophageal adenocarcinoma were labelled for CD3, CD4, CD8, PD-1, PD-L1, FOXP3, CD68, and PanCK to characterise tumour and peri-tumoral immune infiltrate. All patients received the same neoadjuvant chemotherapy prior to oesophagectomy. 14 patients who engaged with a structured prehabilitation exercise program (mixed aerobic and resistance training twice-weekly) were case-matched with 16 control patients who did not. Results The prehabilitation group had a significant increase (per mm2 ) in cytotoxic T-cells (99.4 vs 13.8, p=0.003). There was also a significant increase in immune suppressive macrophages, exhausted T-helper and cytotoxic T-cells. The only significant change in the peritumoural sections was an increase in exhausted CD8 cytotoxic-T cells in the prehabilitation group (3.3 vs 1.4, p=0.043). There was no difference in the number of Treg or T-helper cells within, or around the tumour sections. Conclusions This study is the ‘first-in-man’ to show that exercise prehabilitation during chemotherapy impacts on the tumour immune microenvironment in patients with oesophageal adenocarcinoma. Exercise results in an increased abundance of both anti-tumorigenic and immunosuppressive cell types. These finding warrant further investigation, especially in the context of modern immunotherapy treatments, to understand the clinical impact that exercise oncology has on tumour regression.

Multi-omics analysis-based macrophage differentiation-associated papillary thyroid cancer patient classifier

BackgroundThe reclassification of Papillary Thyroid Carcinoma (PTC) is an area of research that warrants attention. The connection between thyroid cancer, inflammation, and immune responses necessitates considering the mechanisms of differential prognosis of thyroid tumors from an immunological perspective. Given the high adaptability of macrophages to environmental stimuli, focusing on the differentiation characteristics of macrophages might offer a novel approach to address the issues related to PTC subtyping. MethodsSingle-cell RNA sequencing data of medullary cells infiltrated by papillary thyroid carcinoma obtained from public databases was subjected to dimensionality reduction clustering analysis. The RunUMAP and FindAllMarkers functions were utilized to identify the gene expression matrix of different clusters. Cell differentiation trajectory analysis was conducted using the Monocle R package. A complex regulatory network for the classification of Immune status and Macrophage differentiation-associated Papillary Thyroid Cancer Classification (IMPTCC) was constructed through quantitative multi-omics analysis. Immunohistochemistry (IHC) staining was utilized for pathological histology validation. ResultsThrough the integration of single-cell RNA and bulk sequencing data combined with multi-omics analysis, we identified crucial transcription factors, immune cells/immune functions, and signaling pathways. Based on this, regulatory networks for three IMPTCC clusters were established. ConclusionBased on the co-expression network analysis results, we identified three subtypes of IMPTCC: Immune-Suppressive Macrophage differentiation-associated Papillary Thyroid Carcinoma Classification (ISMPTCC), Immune-Neutral Macrophage differentiation-associated Papillary Thyroid Carcinoma Classification (INMPTCC), and Immune-Activated Macrophage differentiation-associated Papillary Thyroid Carcinoma Classification (IAMPTCC). Each subtype exhibits distinct metabolic, immune, and regulatory characteristics corresponding to different states of macrophage differentiation.

Abstract B122: 2 for 1: Targeting tumor-associated macrophages and cancer cells with a novel MERTK-targeting antibody-drug conjugate (ADC)

Abstract MERTK, a member of the TAM family receptor tyrosine kinases, is overexpressed in various solid and hematologic cancers. MERTK signaling drives multiple processes such as proliferation, survival, invasion and drug resistance. Furthermore, MERTK is expressed on M2 tumor-associated macrophages (TAMs), playing a role in the production of immunosuppressive cytokines and efferocytosis. Within normal tissue, MERTK is also expressed in the retinal pigment epithelium (RPE). Our RNA-DRIVErTM platform identified MERTK as a key driver of metastatic progression and thus a potentially potent target for therapeutic intervention. Our MERTK-targeting humanized monoclonal antibody, RGX-019, is internalized by both MERTK-expressing cancer cells and macrophages. In M2 macrophages, RGX-019 induced expression of pro-inflammatory M1 macrophage type cytokines. To effectively target MERTK in both cancer cells and immune-suppressive macrophages, we developed an MMAE antibody drug conjugate (ADC). Here, we assessed the anti-tumor efficacy and retinal safety of RGX-019-MMAE and examined MERTK expression in both solid and hematologic cancers. RGX-019-MMAE induced cytotoxicity against MERTK-expressing cancer cells and mediated bystander anti-tumor efficacy in vitro. In xenograft models, RGX-019-MMAE demonstrated robust anti-tumor efficacy, including complete tumor regressions in various cancers such as TNBC, melanoma, and multiple myeloma. Anti-tumor efficacy of RGX-019-MMAE was associated with improved overall survival. RGX-019-MMAE suppressed syngeneic tumor growth in a mouse model expressing human MERTK extracellular domain, and lacked anti-tumor efficacy in animals not expressing human MERTK. Anti-tumor efficacy was associated with reduced M2 macrophage levels in tumors. Given that RGX-019 only binds human but not murine MERTK, these findings suggest that RGX-019-MMAE targets the host tumor microenvironment to mediate anti-tumor efficacy. Immunohistochemical analysis revealed broad MERTK expression in multiple human cancers with highest expression detected in ovarian, NSCLC, and urothelial cancers. In primary AML samples, significantly higher expression was detected in M5 subtype samples, those with PTPN11 mutations or t(9;11) translocations. Higher MERTK expression in AML correlated with significantly worse patient survival. MERTK in the RPE is involved in maintenance of retinal homeostasis. After a 28-day treatment with RGX-019-MMAE, we observed no signs of retinal degeneration in a transgenic mouse expressing human MERTK extracellular domain. The lack of retinal toxicity can be explained by the partial agonist activity of RGX-019 and absence of MMAE cytotoxicity against non-proliferating RPE cells. The substantial anti-tumor efficacy of RGX-019-MMAE in multiple aggressive tumors, along with the limited expression in normal tissues, reveal MERTK as a promising novel target for an ADC approach. Our findings support the continued advancement of RGX-019-MMAE for the treatment of solid and hematologic cancers through targeting of both TAMs and cancer cells. Citation Format: Shugaku Takeda, PuiChi Lo, Daniel Schefer, Maryam Siddiqui, Anudishi Tyagi, Venkata Lokesh Battula, Abhishek Maiti, Sohail Tavazoie, Masoud Tavazoie, David Martin Darst, Isabel Kurth. 2 for 1: Targeting tumor-associated macrophages and cancer cells with a novel MERTK-targeting antibody-drug conjugate (ADC) [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B122.

CD300f signalling induces inhibitory human monocytes/macrophages

The CD300 glycoproteins are a family of related leucocyte surface molecules that regulate the immune response via their paired triggering and inhibitory receptors. Here we studied CD300f, an apoptotic cell receptor, and how it modulates the function of human monocytes and macrophages. We showed that CD300f signalling by crosslinking with anti-CD300f mAb (DCR-2) suppressed monocytes causing upregulation of the inhibitory molecule, CD274 (PD-L1) and their inhibition of T cell proliferation. Furthermore, CD300f signalling drove macrophages preferentially towards M2-type with upregulation of CD274, which was further enhanced by IL-4. CD300f signalling activates the PI3K/Akt pathway in monocytes. Inhibition of PI3K/Akt signalling resulting from CD300f crosslinking leads to downregulation of CD274 expression on monocytes. These findings highlight the potential use of CD300f blockade in cancer immune therapy to target immune suppressive macrophages in the tumour microenvironment, a known resistance mechanism to PD-1/PD-L1 checkpoint inhibitors.

Investigating Chemokine-Matrix Networks in Breast Cancer: Tenascin-C Sets the Tone for CCL2.

Bidirectional dialogue between cellular and non-cellular components of the tumor microenvironment (TME) drives cancer survival. In the extracellular space, combinations of matrix molecules and soluble mediators provide external cues that dictate the behavior of TME resident cells. Often studied in isolation, integrated cues from complex tissue microenvironments likely function more cohesively. Here, we study the interplay between the matrix molecule tenascin-C (TNC) and chemokine CCL2, both elevated in and associated with the progression of breast cancer and playing key roles in myeloid immune responses. We uncover a correlation between TNC/CCL2 tissue levels in HER2+ breast cancer and examine the physical and functional interactions of these molecules in a murine disease model with tunable TNC levels and in in vitro cellular and cell-free models. TNC supported sustained CCL2 synthesis, with chemokine binding to TNC via two distinct domains. TNC dominated the behavior of tumor-resident myeloid cells; CCL2 did not impact macrophage survival/activation whilst TNC facilitated an immune suppressive macrophage phenotype that was not dependent on or altered by CCL2 co-expression. Together, these data map new binding partners within the TME and demonstrate that whilst the matrix exerts transcriptional control over the chemokine, each plays a distinct role in subverting anti-tumoral immunity.

Single cell RNA sequencing analysis of tumor responses to triple drug oral immunotherapy in dogs with osteosarcoma

Abstract Osteosarcoma constitutes a rare childhood cancer that is associated with dismal outcomes. As such, there is a need to develop non-invasive immunotherapeutic approaches to treat this aggressive malignancy. To address this need, we described the cellular heterogeneity in spontaneously occurring canine osteosarcoma tumors then evaluated how treatment with adjuvant immunotherapy altered the tumor microenvironment. We investigated the effects of a triple drug immunotherapeutic (orally administered losartan, ladarixin, and toceranib) that was designed to deplete immune suppressive macrophages and promote antitumor immunity. The treated cohort of dogs were given the adjuvant immunotherapy for 14 days prior to amputation, while naïve samples were collected from unrelated dogs undergoing amputation to treat a primary osteosarcoma tumor. In total, we completed single cell RNA sequencing on 6 naïve dogs and 6 treated dogs to profile just under 100,000 cells. Unsupervised clustering revealed 30 unique cell populations with T cells, macrophages, and neutrophils making up the majority of immune infiltrates. Evaluation of changes to the cellular proportions following treatment revealed a marked reduction in macrophages, which suggests the combination immunotherapy was able to alter the tumor microenvironment. Overall, this study provides unique insights into the heterogeneity within canine osteosarcoma tumors and highlights the impacts of adjuvant immunotherapy on tumor infiltrating immune cell transcriptional programs. T32 OD012201 U01 CA5341458 Shipley family foundation

Abstract 1798: ALDH inhibition as modulator of ovarian tumor associated immune cells

Abstract Rationale: Ovarian cancer (OC) is an aggressive disease with the 3rd highest mortality to incidence ratio of all cancers. Aldehyde dehydrogenase-1A enzymes play a key role in retinoic acid (RA) synthesis. RA regulates cell proliferation, differentiation, and survival by activating nuclear receptor transcription factors (RAR, RXR). In cancer cells, increased ALDH expression correlates with chemoresistance and poor prognosis. Our goal is to effectively utilize ALDH inhibitors (ALDHi) as novel therapeutics and identify their mechanism in cancer and immune cells. Based on RNAseq data, NR4A1 has emerged as a possible ALDHi target. After binding RXRγ, NR4A1 serves as a ‘molecular switch’ for cancer cell survival. Here, we explore the potential use of ALDHi as immune modulators in OC. Methods: T cells: The effect of several ALDHi on T cells following anti-CD3/CD28 polyclonal stimulation was tested via flow cytometry. Macrophages: Human OC ascites samples were cultured and treated for 72h with ALDHi or DMSO control. Flow cytometry was performed to identify ascites resident cell populations. Western blot to detect NR4A1 was conducted. Dendritic cells (DCs): Murine bone-marrow derived DCs were exposed to ALDHi or DMSO and expanded in culture; cells were counted via hematocytometer to assess myeloid cell expansion and DC differentiation. DC vaccine: ALDHi and DMSO were used with a DC vaccine and sonic hedgehog inhibitor in a study of ID8 tumor-bearing mice to assess effects on tumor progression and survival. Results: We identified several novel ALDHi that promote T cell proliferation and CD8 expansion in vitro. Exposure of 5 patient ascites samples to ALDHi showed a significant decrease in CD14+ and CD163+ cells, indicating a loss of M2 immune-suppressive macrophages vs DMSO. NR4A1 expression was decreased in ALDHi treated cells vs DMSO. In contrast, no deleterious effect was observed on ascites T cells. DC expansion: There was a 10-fold increase in DC proliferation when cells were exposed to ALDHi vs DMSO in culture. DC vaccine: In vivo, addition of ALDHi to a DC vaccine did not provide a survival significance. Discussion: In vitro, ALDHi have differential, complementary effects on tumor and immune cells in OC. In tumor cells, ALDHi lower chemoresistance and trigger necroptosis. ALDHi support the expansion of polyclonal CD8 T cells and type-1 DCs and inhibit T-regulatory cells. In contrast, ALDHi reduce M2 macrophages. Despite beneficial effects in vitro, suggesting anti-tumor competence, there was a modest effect on survival in tumor bearing mice. This may be explained by poor bioavailability of current ALDHi. Further analyses of cryopreserved murine tissues (tumor, lymph nodes, spleen) using flow and CHIP cytometry to assess surface markers, Western blot to test levels of NR4A1, and IHC to assess intratumoral immune cell invasion will continue to test target engagement. Alternative in vivo treatment regimens with other compounds are ongoing. Citation Format: Julia Knight, Bingsi Gao, Mainpal Rana, Ibrahim Uygun, Ronald Buckanovich, Anda Vlad. ALDH inhibition as modulator of ovarian tumor associated immune cells [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 1798.

Abstract 5871: Pan-cancer myeloid cell analysis at the single cell level reveals the influence of distinct organ sites in myeloid cell phenotypes and support targeting S100A4 to reverse immune suppression

Abstract With abundant pro-tumorigenic myeloid cells and few tumoricidal tumor-infiltrating lymphocytes (<5%), GBM is representative of “immune cold” tumors. As such, many different types of immunotherapies have failed to show significant benefits for most glioma patients. Hence, a better understanding of drivers of the immune suppressive microenvironment in GBM and other immune cold tumors is urgently needed to guide future immunotherapy development and application. We recently analyzed 201,986 human glioma and immune cells from 44 tissue fragments from 18 human glioma patients, and present a comprehensive and high-resolution cellular, molecular, and spatial heterogeneity atlas of human glioma. We report an extensive spatial and molecular heterogeneity of glioma and immune cells within the same patient. In addition, we discovered that cell:cell communication between glioma:myeloid cells is considerably more robust than glioma:T-cells, indicating that myeloid cells form a communication hub in vivo. To gain a deeper understanding of these important immune cells, we analyzed 83,479 glioma-infiltrating myeloid cells and identified 9 molecularly distinct myeloid subtypes: 3 microglia subtypes, 3 bone marrow-derived macrophage (BMDM) subtypes, MDSCs, neutrophils, and dendritic cells. Notably, we found that five of these myeloid cell subtype gene signatures are significant predictors of glioma patient survival, independent of glioma cell mutational profiles or gene expression patterns. Leveraging our dataset, we also identified a novel immunotherapy target that is highly expressed in immune-suppressive macrophages and T cells but not in anti-tumor leukocytes: S100A4. We provide both in vitro and in vivo evidence that S100a4 deletion in stromal cells is sufficient to reprogram the immune microenvironment and significantly extend the survival of two independent glioma models. To broaden the potential impact of targeting S100A4 as a selective modulator of immune suppressive leukocytes, we compared the molecular signatures of glioma-associated myeloid cells to those from 12 other cancer types and peripheral blood myeloid cells. We found that S100A4 expression pattern is highly consistent among all tumor types, where its expression is highest in the monocytes and MDSCs and low in most DCs and tissue-resident macrophages. Our preliminary analysis also shows that myeloid cells in gliomas are molecularly distinct from corresponding cell types in other cancers, strongly indicating the role brain microenvironment in influencing the infiltrating BMDM maturation and polarization. Citation Format: Nourhan Abdelfattah, Parveen Kumar, Caiyi Wang, Jia-Shiun Leu, David Baskin, William Flynn, Ruli Gao, Kumar Pichumani, Omkar Ijare, Stephanie Wood, Suzanne Powell, David Haviland, Brittany Parker Kerrigan, Frederick Lang, Sujit Prabhu, Kristin Huntoon, Wen Jiang, Betty Kim, Joshy George, Kyuson Yun. Pan-cancer myeloid cell analysis at the single cell level reveals the influence of distinct organ sites in myeloid cell phenotypes and support targeting S100A4 to reverse immune suppression. [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 5871.

Abstract 5091: Potentiating immunogenicity of PD1 blockage with metronomic Oral CAPOX for local and liver metastasized colorectal cancer

Abstract Since the appearance of oxaliplatin, FOLFOX therapy is positioned as the standard of care for colorectal cancer (CRC). And after the advent of capecitabine, an oral prodrug of 5-FU, both FOLFOX and CAPOX remain the first-line treatment for local and advanced CRC. Even though oral 5-FU prodrug has approved more than two decades ago, orally available oxaliplatin has not been developed yet. Oral chemotherapy has garnered attention in the era of cancer immunotherapy because oral form of the therapy makes metronomic therapy feasible. In this regard, we invented orally absorbable oxaliplatin exploiting complexation with bile acid moiety. The invention encouraged to expand the dosing of combination treatment of 5-FU and oxaliplatin, the most established chemotherapy combination of CRC, into metronomic scheduling. Oral CAPOX therapy is developed to administrate 5-FU and oxaliplatin combination (FOX) metronomically for chemo-immunotherapy. Metronomic dosage successfully induced immunogenic cancer cell death in colorectal cancer xenograft mouse model not exerting toxicity to other organs abolishing immune suppressive effect of chemotherapy. Oral CAPOX boosted tumor immunity translating immune microenvironment. Immune cell population and activation was analyzed using flow cytometry. Suppressive immune cell population decreased after Oral CAPOX treatment. Especially, immune-suppressive macrophage population diminished. Macrophages became immunogenic and presented antigen more frequently. Improved tumor microenvironment combined with anti-PD1 treatment activated and proliferated T cells locally and systemically. Oral CAPOX and anti-PD1 combination therapy activated cytotoxic T cell in tumor tissue and tumor draining lymph node. This therapy also activated NK cells systemically. Oral CAPOX and anti-PD1 combination treatment regressed established tumor completely in more than 90% of the mouse showing its capacity for clinical usage. Liver is the region which makes CRC least immunogenic among metastatic site and the organ CRC is metastasized most abundantly. Immuno-suppresive macrophage in liver is known for the factor discriminating liver metastasized CRC from other organs metastasized one suppressing T cells. Because Oral CAPOX therapy converts macrophage more immunogenic, Oral CAPOX therapy could reverse immunosuppressive tumor microenvironment of liver metastasized colorectal cancer. Oral CAPOX and anti-PD1 combination is treated liver metastasized CRC model and showed standing out antitumor efficacy, and T cell activation. Citation Format: Seong Jin Park, Seho Kweon, Moyo Knowledge Mudhibadhi, Ha Rin Kim, Youngro Byun. Potentiating immunogenicity of PD1 blockage with metronomic Oral CAPOX for local and liver metastasized colorectal cancer. [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 5091.

Editors' Selections from Relevant Scientific Publications

Highlights from the Literature| April 03 2023 Editors' Selections from Relevant Scientific Publications Author & Article Information Online ISSN: 1940-6215 Print ISSN: 1940-6207 ©2023 American Association for Cancer Research2023American Association for Cancer Research Cancer Prev Res (Phila) (2023) 16 (4): 185. https://doi.org/10.1158/1940-6207.CAPR-16-4-HFL Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn MailTo Tools Icon Tools Get Permissions Cite Icon Cite Search Site Article Versions Icon Versions Version of Record April 3 2023 Citation Editors' Selections from Relevant Scientific Publications. Cancer Prev Res (Phila) 1 April 2023; 16 (4): 185. https://doi.org/10.1158/1940-6207.CAPR-16-4-HFL Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest Search Advanced Search Immune cells in colorectal cancer (by Juha P. Väyrynen et al via Cancer Immunology Research) A spatial survey of gene expression and proteomic changes in early-stage colorectal cancer reveals molecular and immunological features during tumorigenesis. Using digital spatial profiling and imaging mass cytometry, Roelands et al. analyzed normal, precancerous, and cancerous tissue samples from patients with early-stage disease and identified differentially expressed genes in the epithelium and stromal segments. They found that innate immune responses were activated early during malignant transformation, and there was a shift of immune cell composition from pro-inflammatory to immune-suppressive macrophage populations from normal tissue through dysplasia to cancer. These findings shed light on the stepwise progression of colorectal cancer and suggest that the approach could aid in identifying biomarkers for early detection and interception. Roelands J, … de Miranda NFCC. Gut. 2022 Nov 28:gutjnl-2022-327608. Small cell lung cancer (by Yale Rosen via Wikimedia Commons)... You do not currently have access to this content.

Abstract IA026: Chromosomal instability as a driver of cancer metastasis

Abstract Chromosomal instability (CIN) is a hallmark of human cancer that is associated with metastasis and immune evasion. Yet, how CIN shapes the tumor microenvironment (TME) to facilitate tumor progression remains poorly understood. We undertook a systems level approach to better define the impact of CIN on cancer cell states as well as TME phenotypes and found that cancer cell CIN engenders a pro-metastatic TME that is markedly enriched for immune-suppressive macrophages, a granulocytic infiltrate, and dysfunctional T cells. We then developed a novel computational tool to infer conditionally dependent cell-cell interactions from single cell RNA sequence data to define tumor-derived factors that shape the TME and promote metastatic dissemination. This work led to the identification of novel therapeutic targets in chromosomally unstable tumors and sheds light on key mechanisms by which CIN cooperates with the immune system to drive disease progression. Citation Format: Samuel F. Bakhoum. Chromosomal instability as a driver of cancer metastasis [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 IA026.

TMIC-63. SELECTIVE TARGETING OF IMMUNE-SUPPRESSIVE LEUKOCYTES TO REPROGRAM THE GBM IMMUNE LANDSCAPE

Abstract A significant barrier to immunotherapy efficacy is GBM’s immunosuppressive microenvironment composed of few tumor-infiltrating lymphocytes (TILs; < 5%) but abundant myeloid cells, making it an “immune cold” tumor. To enhance immunotherapy efficacy, a better molecular and functional understanding of the heterogeneous cell types in the GBM microenvironment and their function is urgently needed. Single cell RNA-sequencing offers high-resolution cellular and molecular data to elucidate cancer and stromal cell phenotypes at the single cell level to identify the most relevant cell types to target to enhance immune activation. We recently reported an integrated, multi-regional and -dimensional single-cell transcriptomic analysis of 201,986 human glioma and immune cells derived from 44 tissue fragments from 18 human glioma patients. In doing so, we mapped GBM cellular heterotypia and spatial, molecular, and functional heterogeneity of glioma associated immune cells. We observed an extensive spatial and molecular heterogeneity of glioma cells, microglia, macrophages, and T cells within the same tumor samples in low-grade gliomas (LGGs), primary GBMs, and recurrent GBMs. Importantly, our analysis of 83,479 glioma infiltrating myeloid cells identifies 9 molecularly distinct myeloid subtypes: 4 microglia, 4 bone marrow-derived macrophage (BMDM), and dendritic cell subtypes. Importantly, five of these myeloid cell subtype gene signatures were independent predictors of patient survival in multiple datasets, demonstrating the importance of immune cells in disease progression and aggressiveness. Here, we report evidence for heterogeneous cell:cell communication between glioma and immune cells in different regions within a patient, using CellChat, Cellphone DB, and NicheNet tools. We also nominate immune modulators other than PD1/PDL1 and CTLA3 as more promising targets in GBM. Finally, we provide evidence that S100A4 is a promising novel immunotherapy target since its high-level expression is required for immune-suppressive macrophages and Tregs to block T cell infiltration and DC maturation.

DKK1 drives immune suppressive phenotypes in intrahepatic cholangiocarcinoma and can be targeted with anti-DKK1 therapeutic DKN-01.

Dickkopf-1 (DKK1) is associated with poor prognosis in intrahepatic cholangiocarcinoma (iCCA), but the mechanisms behind this are unclear. Here, we show that DKK1 plays an immune regulatory role in vivo and inhibition reduces tumour growth. Various in vivo GEMM mouse models and patient samples were utilized to assess the effects of tumour specific DKK1 overexpression in iCCA. DKK1-driven changes to the tumour immune microenvironment were characterized by immunostaining and gene expression analysis. DKK1 overexpressing and damage-induced models of iCCA were used to demonstrate the therapeutic efficacy of DKK1 inhibition in these contexts using the anti-DKK1 therapeutic, DKN-01. DKK1 overexpression in mouse models of iCCA drives an increase in chemokine and cytokine signalling, the recruitment of regulatory macrophages, and promotes the formation of a tolerogenic niche with higher numbers of regulatory T cells. We show a similar association of DKK1 with FOXP3 and regulatory T cells in patient tissue and gene expression data, demonstrating these effects are relevant to human iCCA. Finally, we demonstrate that inhibition of DKK1 with the monoclonal antibody mDKN-01 is effective at reducing tumour burden in two distinct mouse models of the disease. DKK1 promotes tumour immune evasion in iCCA through the recruitment of immune suppressive macrophages. Targeting DKK1 with a neutralizing antibody is effective at reducing tumour growth in vivo. As such, DKK1 targeted and immune modulatory therapies may be an effective strategy in iCCA patients with high DKK1 tumour expression or tolerogenic immune phenotypes.

1678P The interaction between cancer cells and cancer-associated fibroblasts promotes RAS signalling in squamous cell carcinoma, resulting in an immune-suppressive tumour microenvironment and poorer survival outcomes

Cancer-Associated Fibroblasts (CAFs) are a main component of the tumour microenvironment. Recent evidence suggests that high levels of CAFs correlate with poorer prognosis and a pro-tumorigenic microenvironment. The clinical relevance of CAF presence and the mechanisms by which they influence prognosis warrant further research. We used publicly available RNAseq data from The Cancer Genome Atlas (TCGA), gene expression data from cancer-cell (CC)-CAF co-cultures of three different cell lines. Further in vitro validation experiments were performed on human Head and Neck cancer cell lines, patient derived CAFs and macrophages from healthy donors. We generated a gene signature that predicts the abundance of CAF/CC interaction in clinical RNAseq data from a meta-analysis of transcriptomic data derived from Cancer Cell (CC) and CAF co-cultures. This gene signature is predictive of worse overall survival in datasets of Squamous Cell Carcinoma (SCC) patients. CAF abundance and the co-culture signature are strongly correlated with RAS signalling. Mechanistically, we showed that AP-1 transcription factor genes are strongly up-regulated when CCs and CAFs are in close contact. Direct contact between CCs and CAFs upregulates the RAS pathway in both cell types through membrane bound growth factors and growth factor receptors, activating AP-1 activity. We also demonstrated that the RAS / AP-1 axis is used by CCs to induce secretion of pro-tumorigenic and immune-inhibitory cytokines from CAFs, enhancing migration of potentially immune-suppressive macrophages. Of note, patients with high expression of CC-CAF coculture genes show an immune-suppressive microenvironment. Importantly, treatment with ERK inhibitors is able to disrupt the activation of this pathway, blocking cytokine production and impairing macrophage migration. In conclusion, we generated a gene signature predictive of overall survival in SCC patients, linked to cancer cell -CAF interaction and RAS signalling. This study reveals a novel mechanism of crosstalk between CCs and CAFs able to modulate the tumour immune microenvironment.

Abstract 5613: Targeting VSIG4, a novel macrophage checkpoint, repolarizes suppressive macrophages which induces an inflammatory response in primary cell in vitro assays and fresh human tumor cultures, and inhibits tumor growth in in vivo murine tumor models

Abstract VSIG4 is a B7 family related protein with known roles as a complement receptor involved in pathogen clearance as well as a negative regulator of T cell activation by an undetermined mechanism. VSIG4 is expressed in tumor associated macrophages (TAMs) with exquisite specificity. In cancer, increased expression of VSIG4 has been associated with worse survival in multiple indications, including non-small cell lung cancer, multiple myeloma, ovarian cancer, and glioma, suggesting an important role in tumor immune evasion. Based upon computational analysis of transcript data across thousands of primary cancer and normal tissue samples, we hypothesized that VSIG4 has an important regulatory role in promoting M2-like immune suppressive macrophages in the tumor microenvironment, and that targeting VSIG4 via a monoclonal antibody could relieve VSIG4-mediated macrophage suppression by repolarizing TAMs to an inflammatory phenotype capable of coordinating an anti-tumor immune response. The ability of anti-VSIG4 antibodies to repolarize M2-like macrophages and induce broad immune activation was studied in in vitro, ex vivo, and in vivo models. In in vitro primary cell assays, anti-VSIG4 upregulated pro-inflammatory cytokines and chemokines in M-CSF plus IL-10 driven monocyte-derived M2c macrophages, inducing a shift toward an M1-like phenotype. In a multicellular assay system, blockade of VSIG4 on M2c macrophages co-cultured with autologous T cells in the presence of SEB activation upregulated the expression of both pro-inflammatory myeloid-derived cytokines and T cell-derived cytokines, suggesting that macrophage repolarization in vitro can induce T cell activation. To assess these observations in a relevant translational model, fresh, patient-derived tumor samples were treated ex vivo with anti-VSIG4. Across multiple tumor types, anti-VSIG4 treatment resulted in a significant upregulation of cytokines involved in TAM repolarization and T cell activation, and chemokines involved in immune cell recruitment, at levels generally greater than observed by treatment with anti-PD-1. To determine whether targeting VSIG4 can lead to an anti-tumor effect in vivo, syngeneic mouse models were dosed with anti-mouse VSIG4 antibodies. Tumor growth inhibition was observed in syngeneic mouse models dosed with anti-VSIG4 alone and in combination with anti-PD-1. Tumor growth inhibition was partially reversed in studies where CD8+ T cells were depleted, demonstrating that targeting VSIG4 on macrophages can elicit a coordinated attack on tumors in vivo through both innate and adaptive immune responses. Taken together, these data suggest that VSIG4 represents a promising new target capable of stimulating an anti-cancer response via multiple key immune mechanisms. Citation Format: Stephen Sazinsky, Ani Nguyen, Mohammad Zafari, Boris Klebanov, Jessica Ritter, Veronica Komoroski, Denise Manfra, Igor Feldman, Tatiana Novobrantseva. Targeting VSIG4, a novel macrophage checkpoint, repolarizes suppressive macrophages which induces an inflammatory response in primary cell in vitro assays and fresh human tumor cultures, and inhibits tumor growth in in vivo murine tumor models [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 5613.