Myeloid cells are known to suppress antitumour immunity1. However, the molecular drivers of immunosuppressive myeloid cell states are not well defined. Here we used single-cell RNA sequencing of human and mouse non-small cell lung cancer (NSCLC) lesions, and found that in both species the type 2 cytokine interleukin-4 (IL-4) was predicted to be the primary driver of the tumour-infiltrating monocyte-derived macrophage phenotype. Using a panel of conditional knockout mice, we found that only deletion of the IL-4 receptor IL-4Rα in early myeloid progenitors in bone marrow reduced tumour burden, whereas deletion of IL-4Rα in downstream mature myeloid cells had no effect. Mechanistically, IL-4 derived from bone marrow basophils and eosinophils acted on granulocyte-monocyte progenitors to transcriptionally programme the development of immunosuppressive tumour-promoting myeloid cells. Consequentially, depletion of basophils profoundly reduced tumour burden and normalized myelopoiesis. We subsequently initiated a clinical trial of the IL-4Rα blocking antibody dupilumab2-5 given in conjunction with PD-1/PD-L1 checkpoint blockade in patients with relapsed or refractory NSCLC who had progressed on PD-1/PD-L1 blockade alone (ClinicalTrials.gov identifier NCT05013450 ). Dupilumab supplementation reduced circulating monocytes, expanded tumour-infiltrating CD8 T cells, and in one out of six patients, drove a near-complete clinical response two months after treatment. Our study defines a central role for IL-4 in controlling immunosuppressive myelopoiesis in cancer, identifies a novel combination therapy for immune checkpoint blockade in humans, and highlights cancer as a systemic malady that requires therapeutic strategies beyond the primary disease site.

Despite no apparent defects in T cell priming and recruitment to tumors, a large subset of T cell rich tumors fail to respond to immune checkpoint blockade (ICB). We leveraged a neoadjuvant anti-PD-1 trial in patients with hepatocellular carcinoma (HCC), as well as additional samples collected from patients treated off-label, to explore correlates of response to ICB within T cell-rich tumors. We show that ICB response correlated with the clonal expansion of intratumoral CXCL13 + CH25H + IL-21 + PD-1 + CD4 + T helper cells (“CXCL13 + T H ”) and Granzyme K + PD-1 + effector-like CD8 + T cells, whereas terminally exhausted CD39 hi TOX hi PD-1 hi CD8 + T cells dominated in nonresponders. CD4 + and CD8 + T cell clones that expanded post-treatment were found in pretreatment biopsies. Notably, PD-1 + TCF-1 + (Progenitor-exhausted) CD8 + T cells shared clones mainly with effector-like cells in responders or terminally exhausted cells in nonresponders, suggesting that local CD8 + T cell differentiation occurs upon ICB. We found that these Progenitor CD8 + T cells interact with CXCL13 + T H within cellular triads around dendritic cells enriched in maturation and regulatory molecules, or “mregDC”. These results suggest that discrete intratumoral niches that include mregDC and CXCL13 + T H control the differentiation of tumor-specific Progenitor exhasuted CD8 + T cells following ICB.

Peritoneal dialysis (PD) patients are at high risk for peritonitis, an infection of the peritoneum that affects 13% of PD users annually. Relying on subjective peritonitis symptoms results in delayed treatment, leading to high hospitalisation costs, peritoneal scarring, and premature transition to haemodialysis. We have developed and tested a low-cost, easy-to-use technology that uses microscopy and image analysis to screen for peritonitis across the effluent drain tube. Compared to other technologies, our prototype is made from off-the-shelf, low-cost materials. It can be set up quickly and key stakeholders believe it can improve the overall PD experience. We demonstrate that our prototype classifies infection-indicating and healthy white blood cell levels in clinically collected patient effluent with 94% accuracy. Integration of our technology into PD setups as a screening tool for peritonitis would enable earlier physician notification, allowing for prompt diagnosis and treatment to prevent hospitalisations, reduce scarring, and increase PD longevity. Our findings demonstrate the versatility of microscopy and image analysis for infection screening and are a proof of principle for their future applications in health care.

While CRISPR screens are helping uncover genes regulating many cell-intrinsic processes, existing approaches are suboptimal for identifying extracellular gene functions, particularly in the tissue context. Here, we developed an approach for spatial functional genomics called Perturb-map. We applied Perturb-map to knock out dozens of genes in parallel in a mouse model of lung cancer and simultaneously assessed how each knockout influenced tumor growth, histopathology, and immune composition. Moreover, we paired Perturb-map and spatial transcriptomics for unbiased analysis of CRISPR-edited tumors. We found that in Tgfbr2 knockout tumors, the tumor microenvironment (TME) was converted to a fibro-mucinous state, and T cells excluded, concomitant with upregulated TGFβ and TGFβ-mediated fibroblast activation, indicating that TGFβ-receptor loss on cancer cells increased TGFβ bioavailability and its immunosuppressive effects on the TME. These studies establish Perturb-map for functional genomics within the tissue at single-cell resolution with spatial architecture preserved and provide insight into how TGFβ responsiveness of cancer cells can affect the TME.

BACKGROUND:Surgical resection of early stage hepatocellular carcinoma is standard clinical practice; however, most tumours recur despite surgery, and no perioperative intervention has shown a survival benefit. Neoadjuvant immunotherapy has induced pathological responses in multiple tumour types and might decrease the risk of postoperative recurrence in hepatocellular carcinoma. We aimed to evaluate the clinical activity of neoadjuvant cemiplimab (an anti-PD-1) in patients with resectable hepatocellular carcinoma.METHODS:For this single-arm, open-label, phase 2 trial, patients with resectable hepatocellular carcinoma (stage Ib, II, and IIIb) were enrolled and received two cycles of neoadjuvant cemiplimab 350 mg intravenously every 3 weeks followed by surgical resection. Eligible patients were aged 18 years or older, had confirmed resectable hepatocellular carcinoma, an Eastern Cooperative Oncology Group performance status of 0 or 1, and adequate liver function. Patients were excluded if they had metastatic disease, if the surgery was not expected to be curative, if they had a known additional malignancy requiring active treatment, or if they required systemic steroid treatment or any other immunosuppressive therapy. After resection, patients received an additional eight cycles of cemiplimab 350 mg intravenously every 3 weeks in the adjuvant setting. The primary endpoint was significant tumour necrosis on pathological examination (defined as >70% necrosis of the resected tumour). Secondary endpoints included delay of surgery, the proportion of patients with an overall response, change in CD8+ T-cell density, and adverse events. Tumour necrosis and response were analysed in all patients who received at least one dose of cemiplimab and completed surgical resection; safety and other endpoints were analysed in the intention-to-treat population. Patients underwent pre-treatment biopsies and blood collection throughout treatment. This trial is registered with ClinicalTrials.gov (NCT03916627, Cohort B) and is ongoing.FINDINGS:Between Aug 5, 2019, and Nov 25, 2020, 21 patients were enrolled. All patients received neoadjuvant cemiplimab, and 20 patients underwent successful resection. Of the 20 patients with resected tumours, four (20%) had significant tumour necrosis. Three (15%) of 20 patients had a partial response, and all other patients maintained stable disease. 20 (95%) patients had a treatment-emergent adverse event of any grade during the neoadjuvant treatment period. The most common adverse events of any grade were increased aspartate aminotransferase (in four patients), increased blood creatine phosphokinase (in three), constipation (in three), and fatigue (in three). Seven patients had grade 3 adverse events, including increased blood creatine phosphokinase (in two patients) and hypoalbuminaemia (in one). No grade 4 or 5 events were observed. One patient developed pneumonitis, which led to a delay in surgery by 2 weeks.INTERPRETATION:This report is, to our knowledge, the largest clinical trial of a neoadjuvant anti-PD-1 monotherapy reported to date in hepatocellular carcinoma. The observed pathological responses to cemiplimab in this cohort support the design of larger trials to identify the optimal treatment duration and definitively establish the clinical benefit of preoperative PD-1 blockade in patients with hepatocellular carcinoma.FUNDING:Regeneron Pharmaceuticals.

Background Despite advances in therapy, the prognosis of hepatocellular carcinoma (HCC) remains poor. The introduction of biologic drugs, including immune check-point inhibitors, has revolutionized HCC treatment. However, only a portion of patients with HCC respond to immunotherapy and predicting response is an unmet need. In this preliminary study, we assessed the value of quantitative multiparametric MRI (mpMRI) for predicting HCC response to neoadjuvant immunotherapy. Methods In this prospective IRB-approved single-center study, we included 17 patients (M/F 14/3, mean age 65y) with resectable HCC who underwent mpMRI including T1 mapping, 3D MR elastography (MRE), diffusion-weighted imaging (DWI), and dynamic contrast-enhanced (DCE)-MRI with a hepatobiliary contrast agent (gadoxetic acid, Eovist/Primovist, Bayer), at pre-treatment and after completion of anti PD-1 immunotherapy (cemiplimab) as part of a trial. All patients underwent surgical resection. HCC lesions were identified by an experienced radiologist and regions of interest were placed to measure tumor native and post-contrast T1 measured during hepatobiliary phase (T1-HBP), tumor stiffness (TS), apparent diffusion coefficient (ADC), and perfusion parameters. The reference standard was the histopathologic percentage of necrosis. Patients with significant tumor necrosis (STN ≥50%) were considered responders. MRI parameters were compared between responders and non-responders by Mann-Whitney U test, and their diagnostic performance to predict response to treatment was assessed by ROC analysis. Results 17 HCC lesions (6.4±4.9 cm, range 2.0-19.0 cm) were resected in 17 patients. At pre-treatment MRI, tumor native T1 and upslope from DCE-MRI were prolonged in responders compared to non-responders, and predicted response with good diagnostic performance (table 1; T1 AUC(CI)=0.82(0.58-0.99), upslope AUC(CI)=0.80(0.58-0.99). The other parameters had no value in predicting response to immunotherapy (AUC range 0.52-0.68). Tumor native T1 and T1-HBP increased with degree of tumor necrosis at resection (%). Conclusions Our results demonstrate the potential utility of native T1 and upslope measured with DCE-MRI for predicting HCC response to neoadjuvant immunotherapy. These findings require validation in an independent study. Acknowledgements Regeneron Pharmaceuticals, Inc. funded the study. We would like to thank our clinical coordinators, Jordan Cuevas and Jenna Korotkin for helping with patient consent and for maintaining the study database. Enamul H. Bhuiyan gratefully acknowledges Professor Steven Sourbron, The University of Sheffield for his generosity to provide updated PMI software. Trial Registration Neoadjuvant Cemiplimab for the Treatment of Resectable NSCLC, HCC, and HNSCC clinicaltrials.gov identifier NCT03916627 Ethics Approval The study obtained continuing approval from the IRB at Mount Sinai through 01/19/2023, for Regeneron Pharmaceuticals, Inc. Protocol # R2810-ONC-1866/PI: Marron/BRANY File # 19-06-061-05. All participants signed informed consent.

Although it has been more than 2 years since the start of the coronavirus disease 2019 (COVID-19) pandemic, COVID-19 continues to be a worldwide health crisis. Despite the development of preventive vaccines, therapies to treat COVID-19 and other inflammatory diseases remain a major unmet need in medicine. Our study sought to identify drivers of disease severity and mortality to develop tailored immunotherapy strategies to halt disease progression. We assembled the Mount Sinai COVID-19 Biobank, which was composed of almost 600 hospitalized patients followed longitudinally through the peak of the pandemic in 2020. Moderate disease and survival were associated with a stronger antigen presentation and effector T cell signature. In contrast, severe disease and death were associated with an altered antigen presentation signature, increased numbers of inflammatory immature myeloid cells, and extrafollicular activated B cells that have been previously associated with autoantibody formation. In severely ill patients with COVID-19, lung tissue-resident alveolar macrophages not only were drastically depleted but also had an altered antigen presentation signature, which coincided with an influx of inflammatory monocytes and monocyte-derived macrophages. In addition, we found that the size of the alveolar macrophage pool correlated with patient outcome and that alveolar macrophage numbers and functionality were restored to homeostasis in patients who recovered from COVID-19. These data suggest that local and systemic myeloid cell dysregulation are drivers of COVID-19 severity and modulation of alveolar macrophage numbers and activity in the lung may be a viable therapeutic strategy for the treatment of critical inflammatory lung diseases.

Among men, prostate cancer is the second leading cause of cancer-associated mortality, with advanced disease remaining a major clinical challenge. We describe a small molecule, SU086, as a therapeutic strategy for advanced prostate cancer. We demonstrate that SU086 inhibits the growth of prostate cancer cells in vitro, cell-line and patient-derived xenografts in vivo, and ex vivo prostate cancer patient specimens. Furthermore, SU086 in combination with standard of care second-generation anti-androgen therapies displays increased impairment of prostate cancer cell and tumor growth in vitro and in vivo. Cellular thermal shift assay reveals that SU086 binds to heat shock protein 90 (HSP90) and leads to a decrease in HSP90 levels. Proteomic profiling demonstrates that SU086 binds to and decreases HSP90. Metabolomic profiling reveals that SU086 leads to perturbation of glycolysis. Our study identifies SU086 as a treatment for advanced prostate cancer as a single agent or when combined with second-generation anti-androgens.

ABSTRACTHere, we leveraged a large neoadjuvant PD-1 blockade trial in patients with hepatocellular carcinoma (HCC) to search for correlates of response to immune checkpoint blockade (ICB) within T cell-rich tumors. We show that ICB response correlated with the clonal expansion of intratumoral CXCL13+ CH25H+ IL-21+ PD-1+ CD4 T helper cells (CXCL13+ Th) and Granzyme K+ PD-1+ effector-like CD8 T cells, whereas terminally exhausted CD39hi TOXhi PD-1hi CD8 T cells dominated in non-responders. Strikingly, most T cell receptor (TCR) clones that expanded post-treatment were found in pre-treatment biopsies. Notably, PD-1+ TCF-1+ progenitor-like CD8 T cells were present in tumors of responders and non-responders and shared clones mainly with effector-like cells in responders or terminally differentiated cells in non-responders, suggesting that local CD8 T cell differentiation occurs upon ICB. We found that these progenitor CD8 T cells interact with CXCL13+ Th cells within cellular triads around dendritic cells enriched in maturation and regulatory molecules, or “mregDC”. Receptor-ligand analysis revealed unique interactions within these triads that may promote the differentiation of progenitor CD8 T cells into effector-like cells upon ICB. These results suggest that discrete intratumoral niches that include mregDC and CXCL13+ Th cells control the differentiation of tumor-specific progenitor CD8 T cell clones in patients treated with ICB.

Despite their key regulatory role and therapeutic potency, the molecular signatures of interactions between T cells and antigen-presenting myeloid cells within the tumor microenvironment remain poorly characterized. Here, we systematically characterize these interactions using RNA sequencing of physically interacting cells (PIC-seq) and find that CD4+PD-1+CXCL13+ T cells are a major interacting hub with antigen-presenting cells in the tumor microenvironment of human non-small cell lung carcinoma. We define this clonally expanded, tumor-specific and conserved T-cell subset as T-helper tumor (Tht) cells. Reconstitution of Tht cells in vitro and in an ovalbumin-specific αβ TCR CD4+ T-cell mouse model, shows that the Tht program is primed in tumor-draining lymph nodes by dendritic cells presenting tumor antigens, and that their function is important for harnessing the antitumor response of anti-PD-1 treatment. Our molecular and functional findings support the modulation of Tht-dendritic cell interaction checkpoints as a major interventional strategy in immunotherapy.

Abstract Hepatocellular carcinoma (HCC) has a dismal prognosis, and though checkpoint blocking antibodies have significantly improved patient outcome, many patients remain left out highlighting the need to identify additional immune target to enhance therapeutic immunity. Macrophages (MΦ) are an abundant and heterogeneous population in the tumor microenvironment (TME), and are associated with a poor prognosis in multiple tumor types, including HCC, however, their molecular and functional diversity is still poorly understood. We analyzed the molecular and spatial organization patterns of immune cells within the TME and adjacent tissue of 23 resected HCC lesions using single-cell RNA sequencing and multiplex immunohistochemistry (IHC). Strikingly, we found that Kupffer cells (KC), the self-renewing tissue-resident macrophages in liver tissue, are lacking from the TME, which is dominated by monocyte-derived macrophages. ScRNAseq followed by high-resolution clustering identified distinct MΦ molecular programs within the monocyte-derived macrophage compartment. One MΦ subset expressed a shared signature with monocytes including FCN1 and S100A8 in addition to T cell activation genes like CXCL9 and IL32. Conversely, one subset of MΦ expressed FOLR2, SEPP1 and genes of the complement (C1Qs), a program shared with KC in the adjacent uninvolved tissue, and include another intratumoral subset enriched for the expression of TREM2 and GPNMB. Guided by these results, we are developing an IHC antibody panel that allows to visualize distinct MΦ localization in the TME. Intratumoral MΦ interface with, and potentially regulate, the T cell compartment within the TME. We are analyzing HCC tumor lesions in our treatment-naïve cohort and in patients treated with neoadjuvant anti-PD-1 therapy (NCT03916627) to study direct interaction of MΦ and T cells using an innovative technology of physically-interacting cell sequencing (PICseq). This analysis enables us to identify MΦ with direct cell-cell contact with T cells, and our preliminary analysis demonstrates an enrichment in MΦ with a highly immunosuppressive phenotype. We are also using PICseq to map the molecular program of MΦ that are interacting with T cells and that we will corroborated with additional patients. Taken together, our data provide a new understanding of intratumoral MΦ diversity and highlight the presence of specific immunoregulatory MΦ programs unique to tumor lesions, with subsets of these MΦ found to be directly interacting with T cells, potentially modulating anti-tumor responsiveness. Our analysis of resected tumor from anti-PD-1 treated patients, will allow us to correlate MΦ programs, and direct T cell interaction, with clinical response, and will inform therapeutic trials targeting specific MΦ populations so as to improve clinical efficacy of cancer immunotherapy. Citation Format: Pauline Hamon, Assaf Magen, Merav Cohen, Alexandra Tabachnikova, Christie Chang, Mark Buckup, Leanna Troncoso, Zhen Zhao, Joel Kim, Amir Giladi, Nausicaa Malissen, Fiona Desland, Jessica Le Berichel, Ido Amit, Ephraim Kenigsberg, Myron Schwartz, Thomas Marron, Miriam Merad. Characterization of molecular and spatial diversity of macrophages in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 64.

Among men, prostate cancer is the second leading cause of cancer-associated mortality, with advanced disease remaining a major clinical challenge. Chalcones are a major class of widely occurring natural products that are intermediates in plant flavonoid isoflavonoid synthesis. They are characterized by an α,β-unsaturated carbonyl structure with two aromatic rings and commonly act as free-radical scavengers. Herein, we describe a chalcone derivative, SU086, as an anticancer agent for prostate cancer. Proteomic and metabolomic profiling demonstrate that SU086 impairs glycolysis, a critical pathway for cancer growth and survival. SU086 inhibited prostate cancer cell growth, migration, and invasion in vitro. Moreover, SU086 significantly delayed the tumor growth of cell line-derived xenograft models of CRPC as well as patient-derived xenografts (PDXs) in vivo, and the proliferation of primary human prostate cancer patient-derived tissues ex vivo. Furthermore, SU086 strongly synergized with standard of care second-generation anti-androgens, enzalutamide and abiraterone, in inhibiting prostate cancer cell growth in vitro and tumor growth in vivo. Our study identifies SU086 alone or in combination therapy settings as a novel treatment for aggressive prostate cancer. We demonstrate that SU086 represents a highly effective therapeutic strategy for both AR-sensitive and AR-insensitive prostate cancers and may potentially be applicable across multiple cancer types. Citation Format: Tanya Ivanova Stoyanova. Identifying a novel glycolytic inhibitor for treatment of aggressive prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1208.

SUMMARYThe cellular architecture of a tumor, particularly immune composition, has a major impact on cancer outcome, and thus there is an interest in identifying genes that control the tumor microenvironment (TME). While CRISPR screens are helping uncover genes regulating many cell-intrinsic processes, existing approaches are suboptimal for identifying gene functions operating extracellularly or within a tissue context. To address this, we developed an approach for spatial functional genomics called Perturb-map, which utilizes protein barcodes (Pro-Code) to enable spatial detection of barcoded cells within tissue. We show >120 Pro-Codes can be imaged within a tumor, facilitating spatial mapping of 100s of cancer clones. We applied Perturb-map to knockout dozens of genes in parallel in a mouse model of lung cancer and simultaneously assessed how each knockout influenced tumor growth, histopathology, and immune composition. Additionally, we paired Perturb-map and spatial transcriptomics for unbiased molecular analysis of Pro-Code/CRISPR lesions. Our studies found in Tgfbr2 knockout lesions, the TME was converted to a mucinous state and T-cells excluded, which was concomitant with increased TGFβ expression and pathway activation, suggesting Tgfbr2 loss on lung cancer cells enhanced suppressive effects of TGFβ on the TME. These studies establish Perturb-map for functional genomics within a tissue at single cell-resolution with spatial architecture preserved.

Prostate cancer is responsible for over 30,000 US deaths annually, attributed largely to incurable metastatic disease. Here, we demonstrate that high levels of plectin are associated with localized and metastatic human prostate cancer when compared to benign prostate tissues. Knock-down of plectin inhibits prostate cancer cell growth and colony formation in vitro, and growth of prostate cancer xenografts in vivo. Plectin knock-down further impairs aggressive and invasive cellular behavior assessed by migration, invasion, and wound healing in vitro. Consistently, plectin knock-down cells have impaired metastatic colonization to distant sites including liver, lung, kidney, bone, and genitourinary system. Plectin knock-down inhibited number of metastases per organ, as well as decreased overall metastatic burden. To gain insights into the role of plectin in prostate cancer growth and metastasis, we performed proteomic analysis of prostate cancer plectin knock-down xenograft tissues. Gene set enrichment analysis shows an increase in levels of proteins involved with extracellular matrix and laminin interactions, and a decrease in levels of proteins regulating amino acid metabolism, cytoskeletal proteins, and cellular response to stress. Collectively these findings demonstrate that plectin is an important regulator of prostate cancer cell growth and metastasis.