Tumor-infiltrating Leukocytes Research Articles

T-Cell Receptor Repertoire Characteristics Associated with Prognostic Significance in High-Grade Serous Ovarian Carcinoma.

High-grade serous ovarian carcinoma (HGSOC) is a fatal gynecological malignancy. Somatic recombination occurring during T-cell receptor (TCR) development results in TCR diversity, and the TCR repertoire, thus produced, is associated with immune response. This study analyzed the difference in the TCR repertoire and their prognostic significance in 51 patients with HGSOC. The patient's clinical characteristics, gene expression pattern, TCR clonotypes, and degree of tumor-infiltrating leukocytes (TILs) were analyzed, and the patients were divided into groups depending on their recurrence pattern, tumor-infiltrating leukocyte (TIL) score, and homologous recombinant repair pathway deficiency (HRD)-associated mutations. The TCR repertoire was low in patients with recurrence and showed the expansion of eight TCR segments. Interestingly, a few genes correlated with the TCRs also showed a difference in expression according to the prognosis. Among them, seven genes were related to immune responses and KIAA1199 was up-regulated in ovarian cancer. Our study shows that the differences in the TCR repertoire in patients with ovarian cancer and their associated immune pathways could affect the prognosis of HGSOC.

Revisiting targeted therapy and immunotherapy for advanced cholangiocarcinoma.

Cholangiocarcinoma (CCA) is a rare and aggressive type of malignant tumor. In the past few years, there has been an increase in the incidence of CCA. Surgery is the only effective treatment but is only suitable for a small percentage of patients. Comprehensive treatment is the normal therapy for terminal CCA patients, depending basically on gemcitabine and cisplatin combination chemotherapy. In the past decade, the emergence of next-generation sequencing technology can be used for the identification of important molecular features of CCA, and several studies have demonstrated that different CCA subtypes have unique genetic aberrations. Targeting fibroblast growth factor receptor (FGFR), isocitrate dehydrogenase (IDH) and epidermal growth factor receptor 2 (EGFR2) are emerging targeted therapies. In addition, researches have indicated that immunotherapy has a key function in CCA. There is ongoing research on programmed cell death protein 1 inhibitors (PD-1), chimeric antigen receptor T cells (CAR-T) and tumor-infiltrating leukocyte (TILs). Researches have shown that targeted therapy, immunotherapy, and conventional chemotherapy in CCA had certain mechanistic links, and the combination of those can greatly improve the prognosis of advanced CCA patients. This study aimed to review the research progress of targeted therapy and immunotherapy for CCA.

CD25 expression could be a prognostic marker of bexarotene monotherapy for cutaneous T-cell lymphomas.

Bexarotene is often administered to phototherapy-resistant early cutaneous T-cell lymphoma (CTCL) patients as one of the first-line therapies in real-world practice. Since bexarotene reduces the expression of CCR4 in CTCL cells and CCL22 to decrease serum CCL22 levels, bexarotene inhibits the migration of CTCL cells, as well as other CCR4+ cells, such as cytotoxic T cells and regulatory T cells, in the lesional skin of CTCL. In this report, the efficacy of bexarotene in 28 cases of CTCL, as well as its correlations with immunohistochemical profiles of tumour-infiltrating leucocytes (TILs), was retrospectively investigated. The overall response rate at 1 and 4months for the total cohort was 70.8% (95% CI, 50.6%-86.3%) and 47.8% (95% CI, 29.2%-67.0%), respectively. The disease control rate for the total cohort at 4months was 65.2% (95% CI, 44.8%-81.3%). The mean event-free survival for all patients was 4.1months (0.3-68.5months). In addition, the immunoreactive cells were calculated using digital microscopy, suggesting that the ratio of CD25+ cells among TILs was significantly increased in patients who responded to bexarotene (p=0.0209), whereas there were no significant differences in the ratios of CD8+ cells, granulysin+ cells, and Foxp3+ cells among TILs between responder and non-responder patients. Collectively, the ratio of CD25 expression among TILs might be a predictive biomarker for the efficacy of bexarotene.

CEACAM1 Marks Highly Suppressive Intratumoral Regulatory T Cells for Targeted Depletion Therapy.

Regulatory T cells (Tregs) exert immunosuppressive functions and hamper antitumor immune responses in the tumor microenvironment. Understanding the heterogeneity of intratumoral Tregs, and how it changes with tumor progression, will provide clues regarding novel target molecules of Treg-directed therapies. From 42 patients with renal cell carcinoma and 5 patients with ovarian cancer, immune cells from tumor and peripheral blood were isolated. We performed multicolor flow cytometry and RNA-sequencing to characterize the phenotypes and heterogeneity of intratumoral Tregs. In vitro functional assays were performed to evaluate suppressive capacity of Tregs and effect of carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1)-mediated depletion. The CT26 tumor model was used to evaluate the association between intratumoral Tregs and tumor growth, and examine the in vivo role of CEACAM1+ intratumoral Tregs on antitumor immunity. We found that CEACAM1 was selectively expressed on intratumoral Tregs, whereas its expression on peripheral Tregs or other immune cells was low. The CEACAM1+ intratumoral Tregs accumulated with tumor progression, whereas the CEACAM1- subset did not. Notably, we found that CEACAM1 marked intratumoral Tregs that exhibited highly suppressive and activated phenotypes with substantial clonal expansion. Depletion of CEACAM1-expressing cells from tumor-infiltrating leukocytes led to increased effector functions of tumor-infiltrating T cells. Moreover, CEACAM1+ cell depletion further enhanced anti-PD-1-mediated reinvigoration of exhausted CD8+ T cells. CEACAM1 marks highly suppressive subset of intratumoral Tregs, and can be a target for selective depletion of intratumoral Tregs. These results may inform future studies on CEACAM1-mediated depletion in patients with cancer.

Association of KMT2C/D loss-of-function variants with response to immune checkpoint blockades in colorectal cancer.

Immune checkpoint inhibitors (ICIs) have become important treatment strategies, yet responses vary among patients and predictive biomarkers are urgently needed. Mutations in KMT2C and KMT2D lead to increased levels of genomic instability. Therefore, we aimed to examine whether KMT2C/D mutations might be a predictor of immunotherapeutic efficacy. Here, we investigated the associations of KMT2C/D loss-of-function (LOF) variants with tumor mutation burden (TMB), MSI-H, PD-L1 expression, the levels of tumor-infiltrating leukocytes (TILs), and clinical response to ICIs. It was found that KMT2C/D LOF variants were associated with higher TMB. Compared with the non-LOF group, the proportion of patients with MSI-H tumors was larger in the LOF group. PD-L1 expression was higher in the LOF group only for colorectal cancer in both the Chinese and The Cancer Genome Atlas cohorts. Importantly, KMT2C/D LOF variants were associated with decreased regulatory T cells and increased levels of CD8+ T cells, activated NK cells, M1 macrophages, and M2 macrophages in colorectal cancer. However, there was no significant association between KMT2C/D LOF and TILs levels in other cancer types. Consistently, the results showed that KMT2C/D LOF variants were associated with prolonged overall survival only in colorectal cancer (p= 0.0485). We also presented that patients with KMT2C/D LOF mutations exhibited a better clinical response to anti-PD-1 therapy in a Chinese colorectal cancer cohort (p= 0.002). Taken together, these results suggested that KMT2C/D LOF variants could be a useful predictor for ICIs efficacy in colorectal cancer. In addition, the predictive value of KMT2C/D LOF variants was consistent with their association with TILs levels.

Infiltrating CD8+ T cells and M2 macrophages are retained in tumor matrix tracks enriched in low tension fibronectin fibers

Tracks rich in matrix and cells, as described in several cancer types, have immunosuppressive functions and separate tumor nests and stroma, yet their origin is unknown. Immunostainings of cryosections from mouse breast tumors show that these tracks are bordered by an endothelial-like basement membrane, filled with fibers of collagen adjacent to tenascin-C (TNC) and low-tension fibronectin (Fn) fibers. While present in early-stage tumors and maturing with time, tracks still form under TNC KO conditions, however, host (not tumor cell)-derived TNC is important for track maturation. Tumor infiltrating leukocytes (mostly M2 macrophages and CD8+ T cells) are retained in tracks of early-stage tumors. Following track maturation, retained tumor infiltrating leukocyte (TIL) numbers get reduced and more CD8+ TIL enter the tumor nests in the absence of TNC. As these tracks are enriched with platelets and fibrinogen and have a demarcating endothelial-like basement membrane often adjacent to endothelial cells, this suggests a role of blood vessels in the formation of these tracks. The Fn fiber tension probe FnBPA5 colocalizes with TNC and immune cells in the tracks and shows decreased binding in tracks lacking TNC. Consequently, FnBPA5 can serve as probe for tumor matrix tracks that have immune suppressive properties.

Vasoactive intestinal peptide blockade suppresses tumor growth by regulating macrophage polarization and function in CT26 tumor-bearing mice

Macrophages are a major population of immune cells in solid cancers, especially colorectal cancers. Tumor-associated macrophages (TAMs) are commonly divided into M1-like (tumor suppression) and M2-like (tumor promotion) phenotypes. Vasoactive intestinal peptide (VIP) is an immunoregulatory neuropeptide with a potent anti-inflammatory function. Inhibition of VIP signaling has been shown to increase CD8+ T cell proliferation and function in viral infection and lymphoma. However, the role of VIP in macrophage polarization and function in solid tumors remains unknown. Here, we demonstrated that conditioned medium from CT26 (CT26-CM) cells enhanced M2-related marker and VIP receptor (VPAC) gene expression in RAW264.7 macrophages. VIP hybrid, a VIP antagonist, enhanced M1-related genes but reduced Mrc1 gene expression and increased phagocytic ability in CT26-CM-treated RAW264.7 cells. In immunodeficient SCID mice, VIP antagonist alone or in combination with anti-PD-1 antibody attenuated CT26 tumor growth compared with the control. Analysis of tumor-infiltrating leukocytes found that VIP antagonist increased M1/M2 ratios and macrophage phagocytosis of CT26-GFP cells. Furthermore, Vipr2 gene silencing or VPAC2 activation affected the polarization of CT26-CM-treated RAW264.7 cells. In conclusion, the inhibition of VIP signaling enhanced M1 macrophage polarization and macrophage phagocytic function, resulting in tumor regression in a CT26 colon cancer model.

Cell cycle kinases (AUKA, CDK1, PLK1) are prognostic biomarkers and correlated with tumor-infiltrating leukocytes in HBV related HCC

Hepatocellular carcinoma (HCC) is one of the high incidence cancers and third leading cause of cancer-related mortality. HBV is the top most risk factor accounting for 50–80% of the HCC cases. Kinases: Aurora kinase A (AURKA), cyclin-dependent kinase (CDK1) and Polo-like kinase 1 (PLK1), the key regulators of cell mitosis are overexpressed in varieties of cancers including HCC. However, the exact role of these genes in prognosis of HCC is not fully unveiled. In addition, there is no such an accurate prognostic biomarker for HBV-related HCC. To address this issue, we performed a multidimensional analysis of AURKA, CDK1 and PLK1 with a series of publicly available databases in multiple cancers and with experimental validation in HBV-related HCC tissues. Overexpression of AURKA, CDK1 and PLK1 was found in multiple cancers including HCC. Elevated expression of these genes could result from lowered DNA methylation and genomic alterations. Transcriptional overexpression was significantly correlated with poor prognosis of HCC patients. The expression levels were also significantly positively associated with tumor grades and stages. Furthermore, the expression levels of these genes had a strong correlation with infiltration of immune cells. Our analysis shows that AURKA, CDK1 and PLK1 are correlated with immune infiltration and are the prognostic biomarkers for HBV-induced HCC. Communicated by Ramaswamy H. Sarma

Mass cytometry to characterize the immune lung cancer microenvironment.

The human tumor microenvironment requires use of high-dimensional single-cell tools to uncover its cellular complexity and functional variety. For decades, flow cytometry has been the technology of choice to explore immune cell diversity in different pathological contexts. Recently, a new format for flow cytometry - termed mass cytometry - has been developed. It allows for simultaneous interrogation of more than 40 different molecular markers, without the need for spectral compensation, which significantly augments the ability of cytometry to evaluate complex cellular systems and processes. Currently, different multiparametric single-cell analysis approaches are being widely adopted to interrogate the human tumor microenvironment. However, important challenges must be addressed when solid tissues are analyzed by single-cell technologies. This protocol describes the challenge and better use of single-cell mass cytometry to dissect tumor infiltrating leukocytes from surgically resected tumoral lung tissues.

The Lymphatic Endothelium in the Context of Radioimmuno-Oncology

The study of lymphatic tumor vasculature has been gaining interest in the context of cancer immunotherapy. These vessels constitute conduits for immune cells' transit toward the lymph nodes, and they endow tumors with routes to metastasize to the lymph nodes and, from them, toward distant sites. In addition, this vasculature participates in the modulation of the immune response directly through the interaction with tumor-infiltrating leukocytes and indirectly through the secretion of cytokines and chemokines that attract leukocytes and tumor cells. Radiotherapy constitutes the therapeutic option for more than 50% of solid tumors. Besides impacting transformed cells, RT affects stromal cells such as endothelial and immune cells. Mature lymphatic endothelial cells are resistant to RT, but we do not know to what extent RT may affect tumor-aberrant lymphatics. RT compromises lymphatic integrity and functionality, and it is a risk factor to the onset of lymphedema, a condition characterized by deficient lymphatic drainage and compromised tissue homeostasis. This review aims to provide evidence of RT's effects on tumor vessels, particularly on lymphatic endothelial cell physiology and immune properties. We will also explore the therapeutic options available so far to modulate signaling through lymphatic endothelial cell receptors and their repercussions on tumor immune cells in the context of cancer. There is a need for careful consideration of the RT dosage to come to terms with the participation of the lymphatic vasculature in anti-tumor response. Here, we provide new approaches to enhance the contribution of the lymphatic endothelium to radioimmuno-oncology.

Successful targeting of PD-1/PD-L1 with chimeric antigen receptor-natural killer cells and nivolumab in a humanized mouse cancer model.

In recent decades, chimeric antigen receptor (CAR)-engineered immune effector cells have demonstrated promising antileukemic activity. Nevertheless, their efficacy remains unsatisfactory on solid cancers, plausibly due to the influence of tumor microenvironments (TME). In a novel mouse cancer model with a humanized immune system, tumor-infiltrating immunosuppressive leukocytes and exhausted programmed death protein-1 (PD-1)high T cells were found, which better mimic patient TME, allowing the screening and assessment of immune therapeutics. Particularly, membrane-bound programmed death ligand 1 (PD-L1) level was elevated on a tumor cell surface, which serves as an attractive target for natural killer (NK) cell-mediated therapy. Hematopoietic stem cell-derived CAR-NK (CAR pNK) cells targeting the PD-L1 showed enhanced in vitro and in vivo anti-solid tumor function. The CAR pNK cells and nivolumab resulted in a synergistic anti-solid tumor response. Together, our study highlights a robust platform to develop and evaluate the antitumor efficacy and safety of previously unexplored therapeutic regimens.

IMMU-12. ABERRANT EGFR SIGNALING IS ASSOCIATED WITH DIMINISHED CYTOTOXIC T-CELL INFILTRATION IN GLIOBLASTOMA PATIENTS

Abstract Glioblastoma (GBM) has not benefitted from recent advances in immunotherapy compared to other solid malignancies, which suggests that a deeper understanding of the immunobiology of GBM is necessary to develop better therapeutic options. Both oncogenic signaling and evasion of immune destruction are hallmarks of cancer, contributing to tumor growth and reduced survival, respectively. Despite recent evidence in non-CNS malignancies demonstrating the importance of aberrant oncogenic signaling on shaping the tumor immune microenvironment, the impact of aberrant epidermal growth factor (EGFR) signaling – genetically altered in 60% of GBM patients - on the tumor immune microenvironment is not well characterized in GBM. To evaluate the importance of altered EGFR signaling in GBM patient samples, we performed single-cell transcriptomic analysis of primary tumor infiltrating leukocytes (TILs) from twenty-five patients which revealed aberrant EGFR signaling is associated with a unique tumor immune microenvironment in both primary and recurrent disease. Specifically, in the primary disease setting, higher infiltration of macrophages and dendritic cells, and significantly lower infiltration of T-regulatory cells was observed in EGFR altered patients compared to non-altered patients. In recurrent disease, EGFR alterations were associated with significantly diminished infiltration of activated and activated exhausted cytotoxic T-cells compared to unaltered disease. Intriguingly, differential gene expression analysis revealed that EGFR altered patients display significantly increased expression of interferon stimulated genes (ISGs) in both myeloid and lymphoid lineages, while analysis of the tumor compartment unexpectedly revealed greater expression of MHCI genes. Collectively, these data emphasize the impact of aberrant EGFR signaling on the tumor immune microenvironment composition and suggest further characterization of EGFR-immune crosstalk and mechanism is warranted in GBM.

TMIC-51. SEX-SPECIFIC T CELL BEHAVIOR DRIVES DIFFERENTIAL IMMUNE RESPONSES IN GLIOBLASTOMA

Abstract Glioblastoma (GBM) is the most common primary malignant brain tumor and shows poor outcomes as a median survival is 12-18 months with current standard-of-care of therapy. GBM exhibits sex differences in incidence and overall survival, with males experiencing a higher incidence and a worse prognosis compared to females. However, the role of immune cells in GBM sex differences outside of myeloid cells remains poorly understood. Using a syngeneic mouse GBM model, we recapitulated the sex differences observed in patients, with shortened survival in male hosts compared to female hosts. These findings were not recapitulated in immuno-deficient mouse strains such as NSG and RAG1KO mice, suggesting a role for immune system, specifically T cells, in GBM sex differences. Flow cytometry analysis of tumor-infiltrating leukocytes revealed that more T cells were found in female tumors, whereas male tumors were enriched in macrophages. Additionally, more T cells in male tumors exhibited high levels of inhibitory receptors, whereas most female T cells were more functional as measured by expression of anti-tumor cytokines such as TNF, IFN-gamma, and granzyme B. A bone marrow chimera model (BMC) revealed that male T cells retained their phenotype in female hosts, whereas female T cell behavior was affected by the male environment. Yet survival analysis on BMC model and adoptive transfer model suggests strong immune cell-intrinsic effect on controlling tumor progression, further supported by in vitro T cell exhaustion model. Lastly, we found that males have more progenitor exhausted T cells, which led to better response to anti-PD1 treatment. Collectively, these results suggest that both cell-intrinsic and cell-extrinsic factors regulate T cell activity in a sex-specific manner, providing insights to develop sex-specific therapeutic approaches.

Cancer cell-intrinsic XBP1 drives immunosuppressive reprogramming of intratumoral myeloid cells by promoting cholesterol production

A hostile microenvironment in tumor tissues disrupts endoplasmic reticulum homeostasis and induces the unfolded protein response (UPR). A chronic UPR in both cancer cells and tumor-infiltrating leukocytes could facilitate the evasion of immune surveillance. However, how the UPR in cancer cells cripples the anti-tumor immune response is unclear. Here, we demonstrate that, in cancer cells, the UPR component X-box binding protein 1 (XBP1) favors the synthesis and secretion of cholesterol, which activates myeloid-derived suppressor cells (MDSCs) and causes immunosuppression. Cholesterol is delivered in the form of small extracellular vesicles and internalized by MDSCs through macropinocytosis. Genetic or pharmacological depletion of XBP1 or reducing the tumor cholesterol content remarkably decreases MDSC abundance and triggers robust anti-tumor responses. Thus, our data unravel the cell-non-autonomous role of XBP1/cholesterol signaling in the regulation of tumor growth and suggest its inhibition as a useful strategy for improving the efficacy of cancer immunotherapy.

Cabozantinib sensitizes microsatellite stable colorectal cancer to immune checkpoint blockade by immune modulation in human immune system mouse models.

Immune checkpoint inhibitors have been found to be effective in metastatic MSI-high colorectal cancers (CRC), however, have no efficacy in microsatellite stable (MSS) cancers, which comprise the majority of mCRC cases. Cabozantinib is a small molecule multi-tyrosine kinase inhibitor that is FDA approved in advanced renal cell, medullary thyroid, and hepatocellular carcinoma. Using Human Immune System (HIS) mice, we tested the ability of cabozantinib to prime MSS-CRC tumors to enhance the potency of immune checkpoint inhibitor nivolumab. In four independent experiments, we implanted distinct MSS-CRC patient-derived xenografts (PDXs) into the flanks of humanized BALB/c-Rag2nullIl2rγnullSirpαNOD (BRGS) mice that had been engrafted with human hematopoietic stem cells at birth. For each PDX, HIS-mice cohorts were treated with vehicle, nivolumab, cabozantinib, or the combination. In three out of the four models, the combination had a lower tumor growth rate compared to vehicle or nivolumab-treated groups. Furthermore, interrogation of the HIS in immune organs and tumors by flow cytometry revealed increased Granzyme B+, TNFα+ and IFNγ+ CD4+ T cells among the human tumor infiltrating leukocytes (TIL) that correlated with reduced tumor growth in the combination-treated HIS-mice. Notably, slower growth correlated with increased expression of the CD4+ T cell ligand, HLA-DR, on the tumor cells themselves. Finally, the cabozantinib/nivolumab combination was tested in comparison to cobimetinib/atezolizumab. Although both combinations showed tumor growth inhibition, cabozantinib/nivolumab had enhanced cytotoxic IFNγ and TNFα+ T cells. This pre-clinical in vivo data warrants testing the combination in clinical trials for patients with MSS-CRC.

Multimodal single-cell profiling of intrahepatic cholangiocarcinoma defines hyperactivated Tregs as a potential therapeutic target.

The landscape and function of the immune infiltrate of intrahepatic cholangiocarcinoma (iCCA), a rare, yet aggressive tumor of the biliary tract, remains poorly characterized, limiting development of successful immunotherapies. Herein, we aimed to define the molecular characteristics of tumor-infiltrating leukocytes with a special focus on CD4+ regulatory T cells (Tregs). We used high-dimensional single-cell technologies to characterize the T-cell and myeloid compartments of iCCA tissues, comparing these with their tumor-free peritumoral and circulating counterparts. We further used genomics and cellular assays to define the iCCA-specific role of a novel transcription factor, mesenchyme homeobox 1 (MEOX1), in Treg biology. We found poor infiltration of putative tumor-specific CD39+ CD8+ T cells accompanied by abundant infiltration of hyperactivated CD4+ Tregs. Single-cell RNA-sequencing identified an altered network of transcription factors in iCCA-infiltrating compared to peritumoral T cells, suggesting reduced effector functions by tumor-infiltrating CD8+ T cells and enhanced immunosuppression by CD4+ Tregs. Specifically, we found that expression of MEOX1 was highly enriched in tumor-infiltrating Tregs, and demonstrated that MEOX1 overexpression is sufficient to reprogram circulating Tregs to acquire the transcriptional and epigenetic landscape of tumor-infiltrating Tregs. Accordingly, enrichment of the MEOX1-dependent gene program in Tregs was strongly associated with poor prognosis in a large cohort of patients with iCCA. We observed abundant infiltration of hyperactivated CD4+ Tregs in iCCA tumors along with reduced CD8+ T-cell effector functions. Interfering with hyperactivated Tregs should be explored as an approach to enhance antitumor immunity in iCCA. Immune cells have the potential to slow or halt the progression of tumors. However, some tumors, such as intrahepatic cholangiocarcinoma, are associated with very limited immune responses (and infiltration of cancer-targeting immune cells). Herein, we show that a specific population of regulatory T cells (a type of immune cell that actually suppresses the immune response) are hyperactivated in intrahepatic cholangiocarcinoma. Targeting these cells could enable cancer-targeting immune cells to act more effectively and should be looked at as a potential therapeutic approach to this aggressive cancer type.

Phagocytosis Checkpoint Blockade Overcomes Immunosuppression Triggered by Radiation Therapy and PD1 Inhibition

<h3>Purpose/Objective(s)</h3> Trials combining radiation (RT) and anti-PD1 therapy have had largely disappointing results despite promising preclinical data. To better understand this lack of synergy, we used single-cell sequencing (scSeq) in a preclinical mouse model of RT ± anti-PD1 to dissect the dynamics of tumor infiltrating leukocytes (TILs) after treatment. We identified the phagocytic checkpoint SIRPα-CD47 on myeloid cells as a key immunosuppressive pathway and tested the impact of targeting this pathway on combined therapy efficacy. <h3>Materials/Methods</h3> Using a murine model of breast cancer (E0771) we tested the effect of RT (16 Gy x 1 using the SmART+ platform with image guidance) ± anti-PD1± anti-CD47. Tumor growth and Kaplan-Meier survival curves were compared for each treatment group. CD45⁺ TILs were isolated from treated tumors and analyzed by flow cytometry, scSeq, and cellular indexing of transcriptomes (CITEseq) to profile the immune microenvironment and its functional capacity. <h3>Results</h3> First we sequenced 94,503 individual TILs at various timepoints following RT, to date the largest collection of single-cell libraries of the post-RT immune compartment. Most baseline cells were CD11b⁺ macrophages and T cells which were rapidly eliminated by day 3 after RT. The immune compartment was subsequently repopulated by <i>de novo</i> T cells and macrophages at day 7. Several phagocytic checkpoints (SIRPα, LILRB3/4, SLAMF3/7, LRP1) were significantly upregulated on CD11b⁺ macrophages after RT. Given the importance of phagocytic checkpoints in mediating myeloid suppressive function, we tested whether SIRPα-CD47 blockade could enhance anti-PD1/RT activity by alleviating RT-mediated immunosuppression. While anti-PD1/RT and anti-CD47/RT delayed tumor growth versus RT alone, the addition of anti-CD47 to anti-PD1/RT showed the greatest reduction in tumor size and longest survival (P<0.001). To elucidate the mechanisms behind this, we sequenced 16,504 additional cells following RT ± anti-PD1 ± anti-CD47. We identified highly exhausted, M2-like macrophages (CD11b⁺Arg1⁺) in tumors treated with anti-PD1/RT, which were eliminated with the addition of anti-CD47. Moreover, several pro-inflammatory markers (Il1a, Nos2, Mif) and chemokines (CCL3, CXCL1-3) were upregulated with anti-PD1/RT but downregulated with the addition of anti-CD47 despite enhanced efficacy of the triple combination. Closer examination revealed that anti-PD1/RT was associated with neutrophil tumor infiltration that was abrogated with anti-CD47. Lastly addition of anti-CD47 to anti-PD1/RT resulted in dramatic increases in B cell tumor infiltration, which was absent with anti-PD1/RT alone. <h3>Conclusion</h3> SIRPα-CD47 blockade significantly enhances anti-tumor immunity and survival elicited by anti-PD1/RT likely by decreasing neutrophil infiltration and increasing B cell activity. Targeting phagocytic checkpoints may limit myeloid suppression induced by anti-PD1/RT by enhancing B and T cell interactions.

CX3CL1 as potential immunotherapeutic tool for bone metastases in lung cancer: A preclinical study

The chemokine CX3CL1 emerges as a double-edged sword in the pathophysiology of cancer. We investigated whether CX3CL1 would act as an impediment or as a support to the development of non-small cell lung cancer skeletal metastases.We set up an in vivo experimental skeletal metastasis model using the LL2 lung cancer cell line expressing low or high levels of CX3CL1. The resulting bone tumors were analyzed using histological, flow cytometry and transcriptomic techniques.The increased CX3CL1 expression was associated with a strong anti-tumor effect. We observed a significant reduced tumor burden in the high-CX3CL1 group compared to the low-CX3CL1 group with significant differences in the composition of the tumor-infiltrating leukocytes and immunity- and osteogenesis-related gene expression.Our results highlight the CX3CL1 ability to reduce cancer cell tumorigenicity by potentially disrupting both the vicious cycle linking bone resorption and the tumor cell proliferation as well as by generating an immune permissive tumor microenvironment rich in cancer-fighting immune cells, especially M1 monocytes, B cells and NK cells. In that regard, CX3CL1 could be an interesting tool to increase and/or predict the success of immune-based therapies requiring immune cell trafficking.

Galectin-3 as a biomarker in breast neoplasms: Mechanisms and applications in patient care.

Galectin-3 is a member of the lectin family encoded by the LGALS3 gene on chromosome 14. It is secreted by a wide range of immune cells and mammary tumor cells. Through its activity on the tumor microenvironment, in particular on tumor-infiltrating leukocytes, galectin-3 improves the proliferation, survival, and colonizing ability of mammary neoplastic cells. Consequently, galectin-3 expression in the tumor microenvironment could worsen therapeutic outcomes of breast neoplasms and become a biomarker and a therapeutic target in combined immunotherapy in breast neoplasms. There is a limited amount of information that is available on galectin-3 in breast cancer in Africa. In this review, we analyze how galectin-3 influences the tumor microenvironment and its potential as a biomarker and therapeutic target in breast neoplasms. We aim to emphasize the significance of investigating galectin-3 in breast neoplasms in Africa based on the results of studies conducted elsewhere.

OS05.4.A Single-cell characterization of human GBM reveals regional differences in tumor-infiltrating leukocyte activation

Abstract Background Clinical trials of systemic T cell checkpoint blockade in GBM patients showed only disappointing results. This may be attributed in part to the immunosuppressive components of the GBM immune tumor microenvironment (iTME). Therefore, major efforts have been undertaken to describe the GBM iTME on a single cell level. However, human data on the composition of the iTME in different tumor regions (contrast enhancing tumor center versus peripheral infiltration zone) remain scarce. Material and Methods Here, we performed high-depth single-cell RNA sequencing (scRNA-seq) on patient-matched biopsies from tumor center and the peripheral infiltration zone of five primary GBM patients. Additionally, peripheral blood mononuclear cells (PBMC) of the same patients were included in the analysis to explore the transcriptional changes occurring during tumor infiltration of circulating immune cells. Main findings of the transcriptional analysis were confirmed by flow cytometry. Results Through analysis of &amp;gt; 45’000 cells, we revealed a distinct regional transcriptional profile of microglia (MG) and monocyte-derived macrophages (MdM), a non-reactive/exhausted MG subcluster in the GBM iTME and an impaired interferon-response signature in the tumor-peripheral cytotoxic cell compartment. Comparing CD8+ T cells from the tumor periphery to PBMC-derived CD8+ T cells of the same patient revealed CX3CR1high and CX3CR1int CD8+ T cells with effector and memory phenotype, respectively, enriched in the PBMC but lacking in the tumor periphery. Tumor peripheral CD8+ T cells shared features with tissue-resident memory CD8+ T cells with exhausted effector functions. Conclusion Our analysis provides a large-scale dissection of GBM-associated cell types complemented by patient-matched PBMCs, serving as a high dimensional reference map of the human GBM iTME.