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The quality and quantity of tumor-infiltrating lymphocytes, particularly CD8 + T cells, are important parameters for the control of tumor growth and response to immunotherapy. Here, we show in murine and human cancers that these parameters exhibit circadian oscillations, driven by both the endogenous circadian clock of leukocytes and rhythmic leukocyte infiltration, which depends on the circadian clock of endothelial cells in the tumor microenvironment. To harness these rhythms therapeutically, we demonstrate that efficacy of chimeric antigen receptor T cell therapy and immune checkpoint blockade can be improved by adjusting the time of treatment during the day. Furthermore, time-of-day-dependent T cell signatures in murine tumor models predict overall survival in patients with melanoma and correlate with response to anti-PD-1 therapy. Our data demonstrate the functional significance of circadian dynamics in the tumor microenvironment and suggest the importance of leveraging these features for improving future clinical trial design and patient care., Competing Interests: Declaration of interests C.S. has been a consultant for Bayer and received speaker fees from Abbvie. M.J.P. has been a consultant for AstraZeneca, Debiopharm, Elstar Therapeutics, ImmuneOncia, KSQ Therapeutics, MaxiVax, Merck, Molecular Partners, Third Rock Ventures, and Tidal. F.S. received consulting fees from BMS/Celgene, Incyte, Kite/Gilead, speaker fees from Kite/Gilead, Incyte, travel support from Kite/Gilead, Novartis, AstraZeneca, Neovii, Janssen, and research funding from Kite/Gilead, Novartis, and BMS/Celgene. A.D.G. received consulting/advisory/lecture honoraria or research funding from Boehringer Ingelheim, SOTIO, Miltenyi Biotec, Novigenix, and IsoPlexis. R.B. received speaker fees from Janssen and is a mentee of the ENDEAVOUR-Breast program of Daichii Sankyo. All these relationships are unrelated to this study., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Tumor-associated macrophages (TAM) are a diverse population of myeloid cells that are often abundant and immunosuppressive in human cancers. CXCL9 Hi TAM has recently been described to have an antitumor phenotype and is linked to immune checkpoint response. Despite the emerging understanding of the unique antitumor TAM phenotype, there is a lack of TAM-specific therapeutics to exploit this new biological understanding. Here, the discovery and characterization of multiple small-molecule enhancers of chemokine ligand 9 (CXCL9) and their targeted delivery in a TAM-avid systemic nanoformulation is reported. With this strategy, it is efficient encapsulation and release of multiple drug loads that can efficiently induce CXCL9 expression in macrophages, both in vitro and in vivo in a mouse tumor model. These observations provide a window into the molecular features that define TAM-specific states, an insight a novel therapeutic anticancer approach is used to discover., (© 2024 The Authors. Advanced Science published by Wiley‐VCH GmbH.)

The spatial distribution of tumor-infiltrating lymphocytes (TIL) predicts breast cancer outcome and response to systemic therapy, highlighting the importance of an intact tissue structure for characterizing tumors. Here, we present ST-FFPE, a spatial transcriptomics method for the analysis of formalin-fixed paraffin-embedded samples, which opens the possibility of interrogating archival tissue. The method involves extraction, exome capture and sequencing of RNA from different tumor compartments microdissected by laser-capture, and can be used to study the cellular composition of tumor microenvironment. Focusing on triple-negative breast cancer (TNBC), we characterized T cells, B cells, dendritic cells, fibroblasts and endothelial cells in both stromal and intra-epithelial compartments. We found a highly variable spatial distribution of immune cell subsets among tumors. This analysis revealed that the immune repertoires of intra-epithelial T and B cells were consistently less diverse and more clonal than those of stromal T and B cells. T-cell receptor (TCR) sequencing confirmed a reduced diversity and higher clonality of intra-epithelial T cells relative to the corresponding stromal T cells. Analysis of the top 10 dominant clonotypes in the two compartments showed a majority of shared but also some unique clonotypes both in stromal and intra-epithelial T cells. Hyperexpanded clonotypes were more abundant among intra-epithelial than stromal T cells. These findings validate the ST-FFPE method and suggest an accumulation of antigen-specific T cells within tumor core. Because ST-FFPE is applicable for analysis of previously collected tissue samples, it could be useful for rapid assessment of intratumoral cellular heterogeneity in multiple disease and treatment settings., (© 2023 The Authors. International Journal of Cancer published by John Wiley & Sons Ltd on behalf of UICC.)

In cancer patients, dendritic cells (DCs) in tumor-draining lymph nodes can present antigens to naive T cells in ways that break immunological tolerance. The clonally expanded progeny of primed T cells are further regulated by DCs at tumor sites. Intratumoral DCs can both provide survival signals to and drive effector differentiation of incoming T cells, thereby locally enhancing antitumor immunity; however, the paucity of intratumoral DCs or their expression of immunoregulatory molecules often limits antitumor T cell responses. Here, we review the current understanding of DC-T cell interactions at both priming and effector sites of immune responses. We place emerging insights into DC functions in tumor immunity in the context of DC development, ontogeny, and functions in other settings and propose that DCs control at least two T cell-associated checkpoints of the cancer immunity cycle. Our understanding of both checkpoints has implications for the development of new approaches to cancer immunotherapy., Competing Interests: Declaration of interests M.J.P. has served as a consultant for Acthera, AstraZeneca, Debiopharm, Elstar Therapeutics, ImmuneOncia, KSQ Therapeutics, MaxiVax, Merck, Molecular Partners, Siamab Therapeutics, Third Rock Ventures, and Tidal. T.R.M. is a co-founder and consultant, and M.D.P. is a consultant for Monopteros Therapeutics. C.G. has served as a consultant for Cellino Biotech., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Tumor microenvironments (TMEs) influence cancer progression but are complex and often differ between patients. Considering that microenvironment variations may reveal rules governing intratumoral cellular programs and disease outcome, we focused on tumor-to-tumor variation to examine 52 head and neck squamous cell carcinomas. We found that macrophage polarity-defined by CXCL9 and SPP1 (CS) expression but not by conventional M1 and M2 markers-had a noticeably strong prognostic association. CS macrophage polarity also identified a highly coordinated network of either pro- or antitumor variables, which involved each tumor-associated cell type and was spatially organized. We extended these findings to other cancer indications. Overall, these results suggest that, despite their complexity, TMEs coordinate coherent responses that control human cancers and for which CS macrophage polarity is a relevant yet simple variable.

Background: Although immune checkpoint inhibitors have been a breakthrough in clinical oncology, these therapies fail to produce durable responses in a significant fraction of patients. This lack of long-term efficacy may be due to a poor pre-existing network linking innate and adaptive immunity. Here, we present an antisense oligonucleotide (ASO)-based strategy that dually targets toll-like receptor 9 (TLR9) and programmed cell death ligand 1 (PD-L1), aiming to overcome resistance to anti-PD-L1 monoclonal therapy., Methods: We designed a high-affinity immunomodulatory IM-TLR9:PD-L1-ASO antisense oligonucleotide (hereafter, IM-T9P1-ASO) targeting mouse PD-L1 messenger RNA and activating TLR9. Then, we performed in vitro and in vivo studies to validate the IM-T9P1-ASO activity, efficacy, and biological effects in tumors and draining lymph nodes. We also performed intravital imaging to study IM-T9P1-ASO pharmacokinetics in the tumor., Results: IM-T9P1-ASO therapy, unlike PD-L1 antibody therapy, results in durable antitumor responses in multiple mouse cancer models. Mechanistically, IM-T9P1-ASO activates a state of tumor-associated dendritic cells (DCs), referred to here as DC3s, which have potent antitumor potential but express the PD-L1 checkpoint. IM-T9P1-ASO has two roles: it triggers the expansion of DC3s by engaging with TLR9 and downregulates PD-L1, thereby unleashing the antitumor functions of DC3s. This dual action leads to tumor rejection by T cells. The antitumor efficacy of IM-T9P1-ASO depends on the antitumor cytokine interleukin-12 (IL-12), produced by DC3s, and Batf3 , a transcription factor required for DC development., Conclusions: By simultaneously targeting TLR9 and PD-L1, IM-T9P1-ASO amplifies antitumor responses via DC activation, leading to sustained therapeutic efficacy in mice. By highlighting differences and similarities between mouse and human DCs, this study could serve to develop similar therapeutic strategies for patients with cancer., Competing Interests: Competing interests: AZ received consulting/advisor fees from BMS, MSD, Hoffmann–La Roche, NBE Therapeutics, Secarna, ACM Pharma, and Hookipa, and maintains further non-commercial research agreements with Secarna, Hookipa, and Beyondsprings. MP has served as a consultant for Aileron Therapeutics, AstraZeneca, Cygnal Therapeutics, Elstar Therapeutics, ImmuneOncia, KSQ Therapeutics, Merck, Siamab Therapeutics, and Third Rock Ventures. The wife of RB is an employee and shareholder of CSL Behring and RB received speakers fee from Janssen. RHK, JF, AM, and FJ are employed by Secarna. CC is currently employed by Idorsia Pharmaceuticals., (© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Purpose: Adding losartan (LOS) to FOLFIRINOX (FFX) chemotherapy followed by chemoradiation (CRT) resulted in 61% R0 surgical resection in our phase II trial in patients with locally advanced pancreatic cancer (LAPC). Here we identify potential mechanisms of benefit by assessing the effects of neoadjuvant LOS on the tumor microenvironment., Experimental Design: We performed a gene expression and immunofluorescence (IF) analysis using archived surgical samples from patients treated with LOS+FFX+CRT (NCT01821729), FFX+CRT (NCT01591733), or surgery upfront, without any neoadjuvant therapy. We also conducted a longitudinal analysis of multiple biomarkers in the plasma of treated patients., Results: In comparison with FFX+CRT, LOS+FFX+CRT downregulated immunosuppression and pro-invasion genes. Overall survival (OS) was associated with dendritic cell (DC) and antigen presentation genes for patients treated with FFX+CRT, and with immunosuppression and invasion genes or DC- and blood vessel-related genes for those treated with LOS+FFX+CRT. Furthermore, LOS induced specific changes in circulating levels of IL-8, sTie2, and TGF-β. IF revealed significantly less residual disease in lesions treated with LOS+FFX+CRT. Finally, patients with a complete/near complete pathologic response in the LOS+FFX+CRT-treated group had reduced CD4+FOXP3+ regulatory T cells (Tregs), fewer immunosuppressive FOXP3+ cancer cells (C-FOXP3), and increased CD8+ T cells in pancreatic ductal adenocarcinoma lesions., Conclusions: Adding LOS to FFX+CRT reduced pro-invasion and immunosuppression-related genes, which were associated with improved OS in patients with LAPC. Lesions from responders in the LOS+FFX+CRT-treated group had reduced Tregs, decreased C-FOXP3 and increased CD8+ T cells. These findings suggest that LOS may potentiate the benefit of FFX+CRT by reducing immunosuppression., (©2023 American Association for Cancer Research.)

Neutrophils accumulate in solid tumors, and their abundance correlates with poor prognosis. Neutrophils are not homogeneous, however, and could play different roles in cancer therapy. Here, we investigate the role of neutrophils in immunotherapy, leading to tumor control. We show that successful therapies acutely expanded tumor neutrophil numbers. This expansion could be attributed to a Sell hi state rather than to other neutrophils that accelerate tumor progression. Therapy-elicited neutrophils acquired an interferon gene signature, also seen in human patients, and appeared essential for successful therapy, as loss of the interferon-responsive transcription factor IRF1 in neutrophils led to failure of immunotherapy. The neutrophil response depended on key components of anti-tumor immunity, including BATF3-dependent DCs, IL-12, and IFNγ. In addition, we found that a therapy-elicited systemic neutrophil response positively correlated with disease outcome in lung cancer patients. Thus, we establish a crucial role of a neutrophil state in mediating effective cancer therapy., Competing Interests: Declaration of interests M.J.P. has served as consultant for AstraZeneca, Elstar Therapeutics, ImmuneOncia, KSQ Therapeutics, Merck, Siamab Therapeutics, Third Rock Ventures, and Tidal. R.W. has served as a consultant for Moderna, Lumicell, Seer Biosciences, Earli, and Accure Health. The wife of R.B. is an employee and shareholder of CSL Behring, and R.B. received a speaker’s fee from Janssen., (Copyright © 2023 Elsevier Inc. All rights reserved.)

The process of cancer immunosurveillance is a mechanism of tumour suppression that can protect the host from cancer development throughout its lifetime 1,2 . However, it is unknown whether the effectiveness of cancer immunosurveillance fluctuates over a single day. Here we demonstrate that the initial time of day of tumour engraftment dictates the ensuing tumour size across mouse cancer models. Using immunodeficient mice as well as mice lacking lineage-specific circadian functions, we show that dendritic cells (DCs) and CD8 + T cells exert circadian anti-tumour functions that control melanoma volume. Specifically, we find that rhythmic trafficking of DCs to the tumour draining lymph node governs a circadian response of tumour-antigen-specific CD8 + T cells that is dependent on the circadian expression of the co-stimulatory molecule CD80. As a consequence, cancer immunotherapy is more effective when synchronized with DC functions, shows circadian outcomes in mice and suggests similar effects in humans. These data demonstrate that the circadian rhythms of anti-tumour immune components are not only critical for controlling tumour size but can also be of therapeutic relevance., (© 2022. The Author(s).)

Cells in complex organisms undergo frequent functional changes, but few methods allow comprehensive longitudinal profiling of living cells. Here we introduce scission-accelerated fluorophore exchange (SAFE), a method for multiplexed temporospatial imaging of living cells with immunofluorescence. SAFE uses a rapid bioorthogonal click chemistry to remove immunofluorescent signals from the surface of labeled cells, cycling the nanomolar-concentration reagents in seconds and enabling multiple rounds of staining of the same samples. It is non-toxic and functional in both dispersed cells and intact living tissues. We demonstrate multiparameter (n ≥ 14), non-disruptive imaging of murine peripheral blood mononuclear and bone marrow cells to profile cellular differentiation. We also show longitudinal multiplexed imaging of bone marrow progenitor cells as they develop into neutrophils over 6 days and real-time multiplexed cycling of living mouse hepatic tissues. We anticipate that SAFE will find broad utility for investigating physiologic dynamics in living systems., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Background: Single-cell RNA sequencing (scRNA-seq) technologies offer unique opportunities for exploring heterogeneous cell populations. However, in-depth single-cell transcriptomic characterization of complex tissues often requires profiling tens to hundreds of thousands of cells. Such large numbers of cells represent an important hurdle for downstream analyses, interpretation and visualization., Results: We develop a framework called SuperCell to merge highly similar cells into metacells and perform standard scRNA-seq data analyses at the metacell level. Our systematic benchmarking demonstrates that metacells not only preserve but often improve the results of downstream analyses including visualization, clustering, differential expression, cell type annotation, gene correlation, imputation, RNA velocity and data integration. By capitalizing on the redundancy inherent to scRNA-seq data, metacells significantly facilitate and accelerate the construction and interpretation of single-cell atlases, as demonstrated by the integration of 1.46 million cells from COVID-19 patients in less than two hours on a standard desktop., Conclusions: SuperCell is a framework to build and analyze metacells in a way that efficiently preserves the results of scRNA-seq data analyses while significantly accelerating and facilitating them., (© 2022. The Author(s).)

Treatment with checkpoint inhibitors can be extraordinarily effective in a fraction of patients, particularly those whose tumors are pre-infiltrated by T cells. In others, efficacy is considerably lower, which has led to interest in developing strategies for sensitization to immunotherapy. Using various colorectal cancer mouse models, it is shown that the use of Traf2 and Nck-interacting protein kinase inhibitors (TNIKi) unexpectedly increases tumor infiltration by PD-1 + CD8 + T cells, thus contributing to tumor control. This appears to happen by two independent mechanisms, by inducing immunogenic cell death and separately by directly activating CD8. The use of TNIKi achieves complete tumor control in 50% of mice when combined with checkpoint inhibitor targeting PD-1. These findings reveal immunogenic properties of TNIKi and indicate that the proportion of colorectal cancers responding to checkpoint therapy can be increased by combining it with immunogenic kinase inhibitors., (© 2022 The Authors. Advanced Biology published by Wiley-VCH GmbH.)

Neutrophils are the first responders to infection and inflammation and are thus a critical component of innate immune defense. Understanding the behavior of neutrophils as they act within various inflammatory contexts has provided insights into their role in sterile and infectious diseases; however, the field of neutrophils in cancer is comparatively young. Here, we summarize key concepts and current knowledge gaps related to the diverse roles of neutrophils throughout cancer progression. We discuss sources of neutrophil heterogeneity in cancer and provide recommendations on nomenclature for neutrophil states that are distinct in maturation and activation. We address discrepancies in the literature that highlight a need for technical standards that ought to be considered between laboratories. Finally, we review emerging questions in neutrophil biology and innate immunity in cancer. Overall, we emphasize that neutrophils are a more diverse population than previously appreciated and that their role in cancer may present novel unexplored opportunities to treat cancer., (© 2022 Quail et al.)

In the past decade, substantial advances have been made in understanding the biology of tumour-associated macrophages (TAMs), and their clinical relevance is emerging. A particular aspect that is becoming increasingly clear is that the interaction of TAMs with cancer cells and stromal cells in the tumour microenvironment enables and sustains most of the hallmarks of cancer. Therefore, manipulation of TAMs could enable improved disease control in a substantial fraction of patients across a large number of cancer types. In this Review, we examine the diversity of TAMs in various cancer indications and how this heterogeneity is being revisited with the advent of single-cell technologies, and then explore the current knowledge on the functional roles of different TAM states and the prognostic and predictive value of TAM-related signatures. We also review agents targeting TAMs that are currently being or will soon be tested in clinical trials, and how manipulations of TAMs can improve existing anticancer treatments. Finally, we discuss how TAM-targeting approaches could be further integrated into routine clinical practice, considering a precision oncology approach and viewing TAMs as a dynamic population that can evolve under treatment pressure., (© 2022. Springer Nature Limited.)

Background: Secretory carcinoma is a more recently described subtype of salivary gland carcinoma that may pose diagnostic challenges and frequently harbors NTRK fusions that may successfully be targeted by TRK inhibitors in advanced disease., Case: We present the case of a female patient with secretory carcinoma arising in the base of tongue with persistent disease after debulking surgery and definitive chemoradiation. As an alternative to salvage surgery, which would have resulted in significant impairment of swallowing and speech function, a targeted therapy with the TRK-inhibitor larotrectinib against an identified ETV6-NTRK3 fusion product was initiated. Larotrectinib treatment has been well tolerated, resulted in durable complete response and the patient maintains good swallowing and speech function., Conclusion: The presented case underscores the importance of the accurate diagnosis of secretory carcinoma. It further highlights the impact of molecular testing as targeted therapies may play an important role in the management of advanced salivary gland cancers., (© 2021 The Authors. Cancer Reports published by Wiley Periodicals LLC.)

Background: Preclinical studies indicate that the concurrent use of inhibitors of the renin-angiotensin-aldosterone system (RAAS) may improve outcomes in broad groups of patients with cancer. There are limited data on the association between the use of RAAS inhibitors and outcomes among patients treated with immune checkpoint inhibitors (ICIs)., Methods: We performed a retrospective study of all patients treated with an ICI in a single academic network. Of 10,903 patients, 5910 were on any anti-hypertensive medication. Of those on anti-hypertensive therapy, 3426 were prescribed a RAAS inhibitor during ICI treatment, and 2484 were prescribed other anti-hypertensive medications. The primary outcome was overall survival in the entire cohort and in sub-groups by cancer types., Results: Thoracic cancer (34%) and melanoma (16%) were the most common types of cancer. Those prescribed a RAAS inhibitor were older, more frequently male, and had more cardiovascular risk factors. In a Cox proportional hazard model, the concurrent use of RAAS inhibitors was associated with better overall survival (hazard ratio (HR):0.92, [95% Confidence Interval (CI):0.85-0.99], P = .032). Patients with gastrointestinal (HR:0.82, [95% CI: 0.67-1.01], P = .057) and genitourinary cancer (HR:0.81, [95% CI:0.64-1.01], P = .067) had a non-statistically significant better overall survival., Conclusions: In this large retrospective study, patients with hypertension who were concomitantly taking a RAAS inhibitor during ICI therapy had better overall survival. This benefit was primarily noted among patients with gastrointestinal and genitourinary cancers. Prospective randomized trials are warranted to further evaluate and specify the benefit of RAAS inhibitors in patients with cancer who receive ICI therapy., Competing Interests: Conflict of interest statement Dr. Neilan has been a consultant to and received fees from Parexel Imaging, Intrinsic Imaging, H3-Biomedicine, Amgen, Sanofi, Genentech, Roche and AbbVie, outside of the current work. Dr. Neilan also reports consultant fees from Bristol Myers Squibb for a Scientific Advisory Board focused on myocarditis related to immune checkpoint inhibitors and research support from AstraZeneca for work related to immune checkpoint inhibitors. Dr. Sullivan has been a consultant to Asana, AstraZeneca, Bristol Myers Squibb, Eisai, Iovance, Merck, Novartis, Pfizer, Replimune; and received research funding from Amgen and Merck, all outside of the current work. Dr. Jain has received an Honorarium from Amgen; Consultant fees from Chugai, Elpis, Pfizer, SPARC, SynDevRx; Owns equity in Accurius, Enlight, SynDevRx; Served on the Board of Trustees of Tekla Healthcare Investors, Tekla Life Sciences Investors, Tekla Healthcare Opportunities Fund, Tekla World Healthcare Fund and received a research Grant from Boehringer Ingelheim. No funding or reagent from these organizations was used in this study. Dr. Reynolds has received research funding from Project Datasphere and consultant to Teladoc all outside of the current work. Dr. Michielin received fees for advisory roles from BMS, MSD, GSK, Novartis, Roche, Pierre-Fabre and Amgen and research funding from BMS, MSD and Amgen. Other authors have no conflicts of interest or financial disclosure., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Developing strategies to inflame tumors is critical for increasing response to immunotherapy. Here, we report that low-dose radiotherapy (LDRT) of murine tumors promotes T-cell infiltration and enables responsiveness to combinatorial immunotherapy in an IFN-dependent manner. Treatment efficacy relied upon mobilizing both adaptive and innate immunity and depended on both cytotoxic CD4 + and CD8 + T cells. LDRT elicited predominantly CD4 + cells with features of exhausted effector cytotoxic cells, with a subset expressing NKG2D and exhibiting proliferative capacity, as well as a unique subset of activated dendritic cells expressing the NKG2D ligand RAE1. We translated these findings to a phase I clinical trial administering LDRT, low-dose cyclophosphamide, and immune checkpoint blockade to patients with immune-desert tumors. In responsive patients, the combinatorial treatment triggered T-cell infiltration, predominantly of CD4 + cells with Th1 signatures. Our data support the rational combination of LDRT with immunotherapy for effectively treating low T cell-infiltrated tumors. SIGNIFICANCE: Low-dose radiation reprogrammed the tumor microenvironment of tumors with scarce immune infiltration and together with immunotherapy induced simultaneous mobilization of innate and adaptive immunity, predominantly CD4 + effector T cells, to achieve tumor control dependent on NKG2D. The combination induced important responses in patients with metastatic immune-cold tumors. This article is highlighted in the In This Issue feature, p. 1 ., (©2021 The Authors; Published by the American Association for Cancer Research.)

Macrophages often abound within tumors, express colony-stimulating factor 1 receptor (CSF1R), and are linked to adverse patient survival. Drugs blocking CSF1R signaling have been used to suppress tumor-promoting macrophage responses; however, their mechanisms of action remain incompletely understood. Here, we assessed the lung tumor immune microenvironment in mice treated with BLZ945, a prototypical small-molecule CSF1R inhibitor, using single-cell RNA sequencing and mechanistic validation approaches. We showed that tumor control was not caused by CSF1R + cell depletion; instead, CSF1R targeting reshaped the CSF1R + cell landscape, which unlocked cross-talk between antitumoral CSF1R - cells. These cells included IFNγ-producing natural killer and T cells, and an IL12-producing dendritic cell subset, denoted as DC 3 , which were all necessary for CSF1R inhibitor-mediated lung tumor control. These data indicate that CSF1R targeting can activate a cardinal cross-talk between cells that are not macrophages and that are essential to mediate the effects of T cell-targeted immunotherapies and promote antitumor immunity. See related Spotlight by Burrello and de Visser, p. 4., (©2021 American Association for Cancer Research.)

Liver metastasis is a major cause of mortality for patients with colorectal cancer (CRC). Mismatch repair-proficient (pMMR) CRCs make up about 95% of metastatic CRCs, and are unresponsive to immune checkpoint blockade (ICB) therapy. Here we show that mouse models of orthotopic pMMR CRC liver metastasis accurately recapitulate the inefficacy of ICB therapy in patients, whereas the same pMMR CRC tumors are sensitive to ICB therapy when grown subcutaneously. To reveal local, nonmalignant components that determine CRC sensitivity to treatment, we compared the microenvironments of pMMR CRC cells grown as liver metastases and subcutaneous tumors. We found a paucity of both activated T cells and dendritic cells in ICB-treated orthotopic liver metastases, when compared with their subcutaneous tumor counterparts. Furthermore, treatment with Feline McDonough sarcoma (FMS)-like tyrosine kinase 3 ligand (Flt3L) plus ICB therapy increased dendritic cell infiltration into pMMR CRC liver metastases and improved mouse survival. Lastly, we show that human CRC liver metastases and microsatellite stable (MSS) primary CRC have a similar paucity of T cells and dendritic cells. These studies indicate that orthotopic tumor models, but not subcutaneous models, should be used to guide human clinical trials. Our findings also posit dendritic cells as antitumor components that can increase the efficacy of immunotherapies against pMMR CRC., (Copyright © 2021 the Author(s). Published by PNAS.)

Neutrophils have historically been considered a singular, terminally-differentiated cell population, replete with pre-formed granules, poised to react quickly, aggressively, and somewhat non-specifically in the face of a microbial challenge or tissue injury. However, in recent years, neutrophil biologists have started revisiting this simplistic conception. Many studies have identified complexities in neutrophil biology, and these findings have led the field to redefine neutrophil heterogeneity from multiple angles including their development and maturation, their tissue location, and their ability to respond to various (pathological) stimuli. In this review, we discuss the importance of this reassessment within the context of cancer. Experimental evidence supports that neutrophil behavior is diverse, context-dependent, and manipulable; cutting-edge technologies have enabled the identification of neutrophil heterogeneity with high resolution and in an unbiased manner, revealing what may be critical underpinnings of these diverse behaviors, and enabling sophisticated computational assessments of specific programs and interactions. We are coming ever closer to delineating a holistic picture of neutrophil heterogeneity and how it may interplay with cancer stage, tumor microenvironment, and therapy. All of this together paints a promising picture when considering how clinical practice may harness the heterogeneity of these cells, for biomarkers or therapeutic approaches, leveraging what we are learning about these powerful and plentiful immune effectors., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Viruses are the second leading cause of cancer worldwide, and human papillomavirus (HPV)-associated head and neck cancers are increasing in incidence in the United States. HPV preferentially infects the crypts of the tonsils rather than the surface epithelium. The present study sought to characterize the unique microenvironment within the crypts to better understand the viral tropism of HPV to a lymphoid-rich organ. Laser-capture microdissection of distinct anatomic areas (crypts, surface epithelium, and germinal centers) of the tonsil, coupled with transcriptional analysis and multiparameter immunofluorescence staining demonstrated that the tonsillar crypts are enriched with myeloid populations that co-express multiple canonical and noncanonical immune checkpoints, including PD-L1, CTLA-4, HAVCR2 (TIM-3), ADORA2A, IDO1, BTLA, LGALS3, CDH1, CEACAM1, PVR, and C10orf54 (VISTA). The resident monocytes may foster a permissive microenvironment that facilitates HPV infection and persistence. Furthermore, the myeloid populations within HPV-associated tonsil cancers co-express the same immune checkpoints, providing insight into potential novel immunotherapeutic targets for HPV-associated head and neck cancers., (Copyright © 2021 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Purpose: There is increasing effort to discover and integrate predictive and/or prognostic biomarkers into treatment algorithms. While tissue-based methods can reveal tumor-immune cell compositions at a single time point, we propose that single-cell sampling via fine needle aspiration (FNA) can facilitate serial assessment of the tumor immune microenvironment (TME) with a favorable risk-benefit profile., Experimental Design: Primary antibodies directed against 20 murine and 25 human markers of interest were chemically modified via a custom linker-bio-orthogonal quencher (FAST) probe. A FAST-FNA cyclic imaging and analysis pipeline were developed to derive quantitative response scores. Single cells were harvested via FNA and characterized phenotypically and functionally both in preclinical and human samples using the newly developed FAST-FNA assay., Results: FAST-FNA samples analyzed manually versus the newly developed deep learning-assisted pipeline gave highly concordant results. Subsequently, an agreement analysis showed that FAST and flow cytometry of surgically resected tumors were positively correlated with an R 2 = 0.97 in preclinical samples and an R 2 = 0.86 in human samples with the detection of the relevant tumor and immune biomarkers of interest. Finally, the feasibility of applying this minimally invasive approach to analyze the TME during immunotherapy was assessed in patients with cancer revealing local antitumor immune programs., Conclusions: The FAST-FNA is an innovative technology that combines bio-orthogonal chemistry coupled with a computational analysis pipeline for the comprehensive profiling of single cells obtained through FNA. This is the first demonstration that the complex and rapidly evolving TME during treatment can be accurately and serially measured by simple FNA., (©2021 American Association for Cancer Research.)

Cytotoxic T lymphocyte (CTL) responses against tumors are maintained by stem-like memory cells that self-renew but also give rise to effector-like cells. The latter gradually lose their anti-tumor activity and acquire an epigenetically fixed, hypofunctional state, leading to tumor tolerance. Here, we show that the conversion of stem-like into effector-like CTLs involves a major chemotactic reprogramming that includes the upregulation of chemokine receptor CXCR6. This receptor positions effector-like CTLs in a discrete perivascular niche of the tumor stroma that is densely occupied by CCR7 + dendritic cells (DCs) expressing the CXCR6 ligand CXCL16. CCR7 + DCs also express and trans-present the survival cytokine interleukin-15 (IL-15). CXCR6 expression and IL-15 trans-presentation are critical for the survival and local expansion of effector-like CTLs in the tumor microenvironment to maximize their anti-tumor activity before progressing to irreversible dysfunction. These observations reveal a cellular and molecular checkpoint that determines the magnitude and outcome of anti-tumor immune responses., Competing Interests: Declaration of interests S.K. has filed a patent application (PCT/EP2016/074644) related to the use of CXCR6-transduced T cells in tumor therapy. All other authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Immunotherapy is revolutionizing cancer treatment but is often restricted by toxicities. What distinguishes adverse events from concomitant antitumor reactions is poorly understood. Here, using anti-CD40 treatment in mice as a model of T H 1-promoting immunotherapy, we showed that liver macrophages promoted local immune-related adverse events. Mechanistically, tissue-resident Kupffer cells mediated liver toxicity by sensing lymphocyte-derived IFN-γ and subsequently producing IL-12. Conversely, dendritic cells were dispensable for toxicity but drove tumor control. IL-12 and IFN-γ were not toxic themselves but prompted a neutrophil response that determined the severity of tissue damage. We observed activation of similar inflammatory pathways after anti-PD-1 and anti-CTLA-4 immunotherapies in mice and humans. These findings implicated macrophages and neutrophils as mediators and effectors of aberrant inflammation in T H 1-promoting immunotherapy, suggesting distinct mechanisms of toxicity and antitumor immunity., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

The tumor immune microenvironment (TIME) is a complex ecosystem that contains adaptive and innate immune cells that have tumor-promoting and anti-tumor effects. There is still much to learn about the diversity, plasticity, and functions of innate immune cells in the TIME and their roles in determining the response to immunotherapies. Experts discuss recent advances in our understanding of their biology in cancer as well as outstanding questions and potential therapeutic avenues., (Copyright © 2021 Elsevier Inc. All rights reserved.)

There is a need for novel analytical techniques to study the composition of single extracellular vesicles (EV). Such techniques are required to improve the understanding of heterogeneous EV populations, to allow identification of unique subpopulations, and to enable earlier and more sensitive disease detection. Because of the small size of EV and their low protein content, ultrahigh sensitivity technologies are required. Here, an immuno-droplet digital polymerase chain reaction (iddPCR) amplification method is described that allows multiplexed single EV protein profiling. Antibody-DNA conjugates are used to label EV, followed by stochastic microfluidic incorporation of single EV into droplets. In situ PCR with fluorescent reporter probes converts and amplifies the barcode signal for subsequent read-out by droplet imaging. In these proof-of-principle studies, it is shown that multiplex protein analysis is possible in single EV, opening the door for future analyses., (© 2020 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

PD-L1 profiling was recently approved by the US Food and Drug Administration as a companion diagnostic for anti-PD1 treatment in patients with head and neck cancer, ushering in a new era for precision medicine. However, the routine development and implementation of such testing is still limited by current clinical workflows and the lack of better and more comprehensive alternatives. In this review, the authors discuss the real-world challenges of clinically based biomarker testing and highlight the advantages of developing fine-needle aspiration (FNA)-based biomarker testing that would enable frequent and serial tumor sampling. A conceptual and technological innovation is introduced, fast analytical screening technique (FAST)-FNA (FAST chemistry-enabled FNA), which is being developed to inform immunotherapy treatment options in patients with head and neck cancer and to assist with the development of the next generation of predictive biomarkers., (© 2020 American Cancer Society.)

Myeloid cells co-expressing the markers CD11b, Ly-6G, and SiglecF can be found in large numbers in murine lung adenocarcinomas and accelerate cancer growth by fostering tumor cell invasion, angiogenesis, and immunosuppression; however, some of these cells' fundamental features remain unexplored. Here, we show that tumor-infiltrating CD11b + Ly-6G + SiglecF high cells are bona fide mature neutrophils and therefore differ from other myeloid cells, including SiglecF high eosinophils, SiglecF high macrophages, and CD11b + Ly-6G + myeloid-derived suppressor cells. We further show that SiglecF high neutrophils gradually accumulate in growing tumors, where they can live for several days; this lifespan is in marked contrast to that of their SiglecF low counterparts and neutrophils in general, which live for several hours only. Together, these findings reveal distinct attributes for tumor-promoting SiglecF high neutrophils and help explain their deleterious accumulation in the tumor bed., Competing Interests: Declaration of Interests M.J.P. has served as a consultant for Aileron Therapeutics, AstraZeneca, Cygnal Therapeutics, Elstar Therapeutics, KSQ Therapeutics, Merck, and Siamab Therapeutics. A.M.K. is a founder and shareholder in 1CellBio, Inc. These commercial relationships are unrelated to the current study. M.J.P., C.E., and C.P. are inventors on patent application 62/489,118 filed by MGH that covers the detection and targeting of tumor-promoting neutrophils., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Interleukin-12 (IL-12) has emerged as an attractive cytokine for cancer therapy because it has direct anti-cancer effects and additionally plays a critical role in enhancing checkpoint inhibitors. Given these multiple modes of actions, identifying means to pharmacologically induce IL-12 production in the tumor microenvironment has become important. In this review, we highlight therapeutics that promote IL-12 induction in tumor-associated myeloid cells through the non-canonical NFkB pathway. We discuss existing clinical trials and briefly examine the additional pathway targets that warrant further exploration for drug discovery., Competing Interests: RW is a consultant for Tarveda Pharmaceuticals, ModeRNA, Alivio Therapeutics, Lumicell, Accure Health, and Aikili Biosystems. These commercial relationships are unrelated to the current study. MJP is a consultant for AstraZeneca, Elstar Therapeutics, KSQ Therapeutics, Merck, and Siamab Therapeutics. These commercial relationships are unrelated to the current study. PDK: no relevant disclosures., (This journal is © The Royal Society of Chemistry.)

Purpose: Tumors with low frequencies of checkpoint positive tumor-infiltrating lymphocytes (cpTIL) have a low likelihood of response to PD-1 blockade. We conducted a prospective multicenter phase II trial of intratumoral plasmid IL-12 (tavokinogene telseplasmid; "tavo") electroporation combined with pembrolizumab in patients with advanced melanoma with low frequencies of checkpoint positive cytotoxic lymphocytes (cpCTL)., Patients and Methods: Tavo was administered intratumorally days 1, 5, and 8 every 6 weeks while pembrolizumab (200 mg, i.v.) was administered every 3 weeks. The primary endpoint was objective response rate (ORR) by RECIST, secondary endpoints included duration of response, overall survival and progression-free survival. Toxicity was evaluated by the CTCAE v4. Extensive correlative analysis was done., Results: The combination of tavo and pembrolizumab was well tolerated with adverse events similar to those previously reported with pembrolizumab alone. Patients had a 41% ORR ( n = 22, RECIST 1.1) with 36% complete responses. Correlative analysis showed that the combination enhanced immune infiltration and sustained the IL-12/IFNγ feed-forward cycle, driving intratumoral cross-presenting dendritic cell subsets with increased TILs, emerging T cell receptor clones and, ultimately, systemic cellular immune responses., Conclusions: The combination of tavo and pembrolizumab was associated with a higher than expected response rate in this poorly immunogenic population. No new or unexpected toxicities were observed. Correlative analysis showed T cell infiltration with enhanced immunity paralleling the clinical activity in low cpCTL tumors., (©2020 American Association for Cancer Research.)

The coronavirus disease 2019 (COVID-19) pandemic has highlighted the need for different types of diagnostics, comparative validation of new tests, faster approval by federal agencies, and rapid production of test kits to meet global demands. In this Perspective, we discuss the utility and challenges of current diagnostics for COVID-19., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Neutrophils are an essential part of the innate immune system. To study their importance, experimental studies often aim to deplete these cells, generally by injecting anti-Ly6G or anti-Gr1 antibodies. However, these approaches are only partially effective, transient or lack specificity. Here we report that neutrophils remaining after anti-Ly6G treatment are newly derived from the bone marrow, instead of depletion escapees. Mechanistically, newly generated, circulating neutrophils have lower Ly6G membrane expression, and consequently reduced targets for anti-Ly6G-mediated depletion. To overcome this limitation, we develop a double antibody-based depletion strategy that enhances neutrophil elimination by anti-Ly6G treatment. This approach achieves specific, durable and controlled reduction of neutrophils in vivo, and may be suitable for studying neutrophil function in experimental models.

Tumour-infiltrating myeloid cells (TIMCs) are critical regulators of cancer growth. The different phenotypes, functions and therapeutic effects of these phagocytes have, however, been difficult to study. With the advent of single-cell-based technologies, a new 'worldview' is emerging: the classification of TIMCs into subtypes that are conserved across patients and across species. As the landscape of TIMCs is beginning to be understood, it opens up questions about the function of each TIMC subtype and its drugability. In this Perspective, we outline the current map of TIMC populations in cancer and their known and presumed functions, and discuss their therapeutic implications and the biological research questions that they give rise to. The answers should be particularly relevant for bioengineers, materials scientists and the chemical and pharmaceutical communities developing the next generation of cancer therapies.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The spleen is a large lymphoid organ located in the abdomen that filters blood and regulates the immune system. The extent of mobilization of splenic immune cells to peripheral tissues in health and disease, however, remains poorly understood. This is due, in large part, to a lack of in vivo, spleen-specific lineage tagging strategies. Here, we describe a detailed practical protocol of spleen transplantation and its evaluation for long-term graft survival. Unlike implantation of splenic morsels in the great omentum, our approach uses arterial and venous anastomoses which rapidly restores blood flow and facilitates long-term survival of the graft. The use of congenic mouse strains permits the use of immunofluorescence and flow cytometry-based methodologies to unambiguously track the migration of spleen-derived cells to peripheral tissues.

Immune-checkpoint blockers can promote sustained clinical responses in a subset of cancer patients. Recent research has shown that a subpopulation of tumor-infiltrating dendritic cells functions as gatekeepers, sensitizing tumors to anti-PD-1 treatment via production of interleukin-12 (IL-12). Hypothesizing that myeloid cell-targeted nanomaterials could be used to deliver small-molecule IL-12 inducers, we performed high-content image-based screening to identify the most efficacious small-molecule compounds. Using one lead candidate, LCL161, we created a myeloid-targeted nanoformulation that induced IL-12 production in intratumoral myeloid cells in vivo, slowed tumor growth as a monotherapy, and had no significant systemic toxicity. These results pave the way for developing combination immunotherapeutics by harnessing IL-12 production for immunostimulation., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Tumor-associated macrophages (TAMs) are often abundant in solid cancers, assuming an immunosuppressive (M2-like) phenotype which supports tumor growth and immune escape. Recent methods have focused on identification of means (e.g., drugs, nanomaterials) that polarize TAMs to a tumor suppressive (M1-like) phenotype; however, reducing the systemic side effects of these therapies and enabling their delivery to TAMs has remained a challenge. Methods: Here, we develop R848-Ad, an adamantane-modified derivative of the toll-like receptor (TLR) 7/8 agonist resiquimod (R848) through iterative drug screening against reporter cell lines. The adamantane undergoes guest-host interaction with cyclodextrin nanoparticles (CDNPs), enabling drug loading under aqueous conditions and TAM-targeted drug delivery. Therapeutic efficacy and systemic side effects were examined in a murine MC38 cancer model. Results: R848-Ad retained macrophage polarizing activity through agonization of TLR7/8, and the adamantane moiety improved drug affinity for the CDNP. In preclinical studies, nanoformulated R848-Ad resulted in a drastic reduction in measurable systemic effects (loss of body weight) relative to similarly formulated R848 alone while arresting tumor growth. Conclusions: The findings demonstrate the ability of strong nanoparticle-drug interactions to limit systemic toxicity of TLR agonists while simultaneously maintaining therapeutic efficacy., Competing Interests: Competing Interests: C.B.R. and R.W. are listed on a patent filed by Partners Healthcare. The remaining authors declare no competing interests., (© The author(s).)

Myocardial infarction, stroke, and sepsis trigger systemic inflammation and organism-wide complications that are difficult to manage. Here, we examined the contribution of macrophages residing in vital organs to the systemic response after these injuries. We generated a comprehensive catalog of changes in macrophage number, origin, and gene expression in the heart, brain, liver, kidney, and lung of mice with myocardial infarction, stroke, or sepsis. Predominantly fueled by heightened local proliferation, tissue macrophage numbers increased systemically. Macrophages in the same organ responded similarly to different injuries by altering expression of tissue-specific gene sets. Preceding myocardial infarction improved survival of subsequent pneumonia due to enhanced bacterial clearance, which was caused by IFNɣ priming of alveolar macrophages. Conversely, EGF receptor signaling in macrophages exacerbated inflammatory lung injury. Our data suggest that local injury activates macrophages in remote organs and that targeting macrophages could improve resilience against systemic complications following myocardial infarction, stroke, and sepsis., (Copyright © 2019 Elsevier Inc. All rights reserved.)

LTX-315 is an oncolytic peptide that has antitumor efficacy in mice grafted with various tumor cell lines and is currently being tested in phase II clinical trials. Here we aimed to further evaluate LTX-315 in conditional genetic mouse models of cancer that typically resist current treatment options and to better understand the drug's mode of action in vivo . We report LTX-315 mediates profound antitumor effects against Braf- and Pten -driven melanoma and delays the progression of Kras- and P53- driven soft tissue sarcoma in mice. Additionally, we show in melanoma that LTX-315 triggers two sequential phases of antitumor response. The first phase of response, which begins within minutes of drug delivery into tumors, is defined by disrupted tumor vasculature and decreased tumor burden and occurs independently of lymphocytes. The second phase of response, which continues over weeks, is defined by long-term alteration of the tumor microenvironment; the changes induced by LTX-315 are most notably characterized by CD8+ T cell infiltration. We further show that these CD8+ T cells are involved in suppressing melanoma outgrowth in mice and report similar CD8+ T cell infiltration following LTX-315 treatment in melanoma and sarcoma patients. Taken together, these findings reveal LTX-315's multiple antitumor effects, including disrupting the tumor vasculature and promoting the conversion of poorly immunogenic tumors into ones that display antitumor T cell immunity., Competing Interests: Conflict of interest: This project was supported by Lytix Biopharma. M.J.P. has served as a consultant for Baxalta, Deciphera Pharmaceuticals, FORMA Therapeutics, Incyte Pharmaceuticals, Jounce Therapeutics, KSQ Therapeutics, Secarna, Siamab, and Syndax; these commercial relationships are unrelated to the current study. R.W. is a cofounder of T2 Biosystems, Lumicell, Accure Health, and VisEn Medical (acquired by Perkin Elmer) and advises Moderna, Alivio Therapeutics, Tarveda Therapeutics, and BioAnalytix; these relationships are unrelated to the current study., (Copyright: © 2019 Liao et al.)

Tumor-infiltrating myeloid cells (TIMs) comprise monocytes, macrophages, dendritic cells, and neutrophils, and have emerged as key regulators of cancer growth. These cells can diversify into a spectrum of states, which might promote or limit tumor outgrowth but remain poorly understood. Here, we used single-cell RNA sequencing (scRNA-seq) to map TIMs in non-small-cell lung cancer patients. We uncovered 25 TIM states, most of which were reproducibly found across patients. To facilitate translational research of these populations, we also profiled TIMs in mice. In comparing TIMs across species, we identified a near-complete congruence of population structures among dendritic cells and monocytes; conserved neutrophil subsets; and species differences among macrophages. By contrast, myeloid cell population structures in patients' blood showed limited overlap with those of TIMs. This study determines the lung TIM landscape and sets the stage for future investigations into the potential of TIMs as immunotherapy targets., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Rationale: After a stroke, patients frequently experience altered systemic immunity resulting in peripheral immunosuppression and higher susceptibility to infections, which is at least partly attributed to lymphopenia. The mechanisms that profoundly change the systemic leukocyte repertoire after stroke are incompletely understood. Emerging evidence indicates that stroke alters hematopoietic output of the bone marrow., Objective: To explore the mechanisms that lead to defects of B lymphopoiesis after ischemic stroke., Methods and Results: We here report that ischemic stroke triggers brain-bone marrow communication via hormonal long-range signals that regulate hematopoietic B lineage decisions. Bone marrow fluorescence-activated cell sorter analyses and serial intravital microscopy indicate that transient middle cerebral artery occlusion in mice arrests B-cell development beginning at the pro-B-cell stage. This phenotype was not rescued in Myd88 -/- and TLR4 -/- mice with disrupted TLR (Toll-like receptor) signaling or after blockage of peripheral sympathetic nerves. Mechanistically, we identified stroke-induced glucocorticoid release as the main instigator of B lymphopoiesis defects. B-cell lineage-specific deletion of the GR (glucocorticoid receptor) in CD19-Cre loxP Nr3c1 mice attenuated lymphocytopenia after transient middle cerebral artery. In 20 patients with acute stroke, increased cortisol levels inversely correlated with blood lymphocyte numbers., Conclusions: Our data demonstrate that the hypothalamic-pituitary-adrenal axis mediates B lymphopoiesis defects after ischemic stroke.