Cytokine-armed dendritic cell progenitors for antigen-agnostic cancer immunotherapy.

Dendritic cells (DCs) are antigen-presenting myeloid cells that regulate T cell activation, trafficking and function. Monocyte-derived DCs pulsed with tumor antigens have been tested extensively for therapeutic vaccination in cancer, with mixed clinical results. Here, we present a cell-therapy platform based on mouse or human DC progenitors (DCPs) engineered to produce two immunostimulatory cytokines, IL-12 and FLT3L. Cytokine-armed DCPs differentiated into conventional type-I DCs (cDC1) and suppressed tumor growth, including melanoma and autochthonous liver models, without the need for antigen loading or myeloablative host conditioning. Tumor response involved synergy between IL-12 and FLT3L and was associated with natural killer and T cell infiltration and activation, M1-like macrophage programming and ischemic tumor necrosis. Antitumor immunity was dependent on endogenous cDC1 expansion and interferon-γ signaling but did not require CD8+ T cell cytotoxicity. Cytokine-armed DCPs synergized effectively with anti-GD2 chimeric-antigen receptor (CAR) T cells in eradicating intracranial gliomas in mice, illustrating their potential in combination therapies.

HTATIP2 regulates arteriogenic activity in monocytes from patients with limb ischemia.

Use of autologous cells isolated from elderly patients with multiple comorbidities may account for the modest efficacy of cell therapy in patients with chronic limb threatening ischemia (CLTI). We aimed to determine whether proarteriogenic monocyte/macrophages (Mo/MΦs) from patients with CLTI were functionally impaired and to demonstrate the mechanisms related to any impairment. Proarteriogenic Mo/MΦs isolated from patients with CLTI were found to have an impaired capacity to promote neovascularization in vitro and in vivo compared with those isolated from healthy controls. This was associated with increased expression of human HIV-1 TAT interactive protein-2 (HTATIP2), a transcription factor known to suppress angiogenesis/arteriogenesis. Silencing HTATIP2 restored the functional capacity of CLTI Mo/MΦs, which was associated with increased expression of arteriogenic regulators Neuropilin-1 and Angiopoietin-1, and their ability to enhance angiogenic (endothelial tubule formation) and arteriogenic (smooth muscle proliferation) processes in vitro. In support of the translational relevance of our findings, silencing HTATIP2 in proarteriogenic Mo/MΦs isolated from patients with CLTI rescued their capacity to enhance limb perfusion in the ischemic hindlimb by effecting greater angiogenesis and arteriogenesis. Ex vivo modulation of HTATIP2 may offer a strategy for rescuing the functional impairment of pro-angio/arteriogenic Mo/MΦs prior to autologous delivery and increase the likelihood of clinical efficacy.

Exosomes, microvesicles, and other extracellular vesicles-a Keystone Symposia report.

Extracellular vesicles (EVs) are small, lipid-bilayer-bound particles released by cells that can contain important bioactive molecules, including lipids, RNAs, and proteins. Once released in the extracellular environment, EVs can act as messengers locally as well as to distant tissues to coordinate tissue homeostasis and systemic responses. There is a growing interest in not only understanding the physiology of EVs as signaling particles but also leveraging them as minimally invasive diagnostic and prognostic biomarkers (e.g., they can be found in biofluids) and drug-delivery vehicles. On October 30-November 2, 2022, researchers in the EV field convened for the Keystone symposium "Exosomes, Microvesicles, and Other Extracellular Vesicles" to discuss developing standardized language and methodology, new data on the basic biology of EVs and potential clinical utility, as well as novel technologies to isolate and characterize EVs.

Defining and targeting tumor-associated macrophages in malignant mesothelioma.

Defining the ontogeny of tumor-associated macrophages (TAM) is important to develop therapeutic targets for mesothelioma. We identified two distinct macrophage populations in mouse peritoneal and pleural cavities, the monocyte-derived, small peritoneal/pleural macrophages (SPM), and the tissue-resident large peritoneal/pleural macrophages (LPM). SPM rapidly increased in tumor microenvironment after tumor challenge and contributed to the vast majority of M2-like TAM. The selective depletion of M2-like TAM by conditional deletion of the Dicer1 gene in myeloid cells (D-/-) promoted tumor rejection. Sorted SPM M2-like TAM initiated tumorigenesis in vivo and in vitro, confirming their capacity to support tumor development. The transcriptomic and single-cell RNA sequencing analysis demonstrated that both SPM and LPM contributed to the tumor microenvironment by promoting the IL-2-STAT5 signaling pathway, inflammation, and epithelial-mesenchymal transition. However, while SPM preferentially activated the KRAS and TNF-α/NFkB signaling pathways, LPM activated the IFN-γ response. The importance of LPM in the immune response was confirmed by depleting LPM with intrapleural clodronate liposomes, which abrogated the antitumoral memory immunity. SPM gene signature could be identified in pleural effusion and tumor from patients with untreated mesothelioma. Five genes, TREM2, STAB1, LAIR1, GPNMB, and MARCO, could potentially be specific therapeutic targets. Accordingly, Trem2 gene deletion led to reduced SPM M2-like TAM with compensatory increase in LPM and slower tumor growth. Overall, these experiments demonstrate that SPM M2-like TAM play a key role in mesothelioma development, while LPM more specifically contribute to the immune response. Therefore, selective targeting of monocyte-derived TAM may enhance antitumor immunity through compensatory expansion of tissue-resident TAM.

Dendritic cell cross-dressing and tumor immunity.

In addition to direct and cross-presentation, dendritic cells (DCs) can present tumor antigens (TAs) to T cells via a hitherto poorly understood mechanism called "cross-dressing." DC cross-dressing involves the acquisition of preformed peptide-major histocompatibility class I/II (p-MHC) complexes from cancer cells. This process has been documented both in cell culture and in tumor models; may occur via the uptake of tumor-derived extracellular vesicles or the horizontal transfer of plasma membrane fragments from cancer cells to DCs; and can be enhanced through DC engineering for therapeutic applications. In some experimental contexts, DC cross-dressing may be essential for productive anti-tumor immunity, possibly owing to the fact that tumor-derived p-MHC complexes encompass the full repertoire of immunologically relevant TAs against which primed cytotoxic T cells can exert their tumoricidal activity.

Apelin-driven endothelial cell migration sustains intestinal progenitor cells and tumor growth.

Stem and progenitor cells residing in the intestinal crypts drive the majority of colorectal cancers (CRCs), yet vascular contribution to this niche remains largely unexplored. VEGFA is a key driver of physiological and tumor angiogenesis. Accordingly, current anti-angiogenic cancer therapies target the VEGFA pathway. Here we report that in CRC expansion of the stem/progenitor pool in intestinal crypts requires VEGFA-independent growth and remodeling of blood vessels. Epithelial transformation induced expression of the endothelial peptide apelin, directs migration of distant venous endothelial cells towards progenitor niche vessels ensuring optimal perfusion. In the absence of apelin, loss of injury-inducible PROX1+ epithelial progenitors inhibited both incipient and advanced intestinal tumor growth. Our results establish fundamental principles for the reciprocal communication between vasculature and the intestinal progenitor niche and provide a mechanism for resistance to VEGFA-targeting drugs in CRCs.

The angiopoietin-Tie2 pathway regulates Purkinje cell dendritic morphogenesis in a cell-autonomous manner.

Neuro-vascular communication is essential to synchronize central nervous system development. Here, we identify angiopoietin/Tie2 as a neuro-vascular signaling axis involved in regulating dendritic morphogenesis of Purkinje cells (PCs). We show that in the developing cerebellum Tie2 expression is not restricted to blood vessels, but it is also present in PCs. Its ligands angiopoietin-1 (Ang1) and angiopoietin-2 (Ang2) are expressed in neural cells and endothelial cells (ECs), respectively. PC-specific deletion of Tie2 results in reduced dendritic arborization, which is recapitulated in neural-specific Ang1-knockout and Ang2 full-knockout mice. Mechanistically, RNA sequencing reveals that Tie2-deficient PCs present alterations in gene expression of multiple genes involved in cytoskeleton organization, dendritic formation, growth, and branching. Functionally, mice with deletion of Tie2 in PCs present alterations in PC network functionality. Altogether, our data propose Ang/Tie2 signaling as a mediator of intercellular communication between neural cells, ECs, and PCs, required for proper PC dendritic morphogenesis and function.

Overcoming microenvironmental resistance to PD-1 blockade in genetically engineered lung cancer models.

Immune checkpoint blockade (ICB) with PD-1 or PD-L1 antibodies has been approved for the treatment of non-small cell lung cancer (NSCLC). However, only a minority of patients respond, and sustained remissions are rare. Both chemotherapy and antiangiogenic drugs may improve the efficacy of ICB in mouse tumor models and patients with cancer. Here, we used genetically engineered mouse models of Kras G12D/+;p53 -/- NSCLC, including a mismatch repair-deficient variant (Kras G12D/+;p53 -/-;Msh2 -/-) with higher mutational burden, and longitudinal imaging to study tumor response and resistance to combinations of ICB, antiangiogenic therapy, and chemotherapy. Antiangiogenic blockade of vascular endothelial growth factor A and angiopoietin-2 markedly slowed progression of autochthonous lung tumors, but contrary to findings in other cancer types, addition of a PD-1 or PD-L1 antibody was not beneficial and even accelerated progression of a fraction of the tumors. We found that antiangiogenic treatment facilitated tumor infiltration by PD-1+ regulatory T cells (Tregs), which were more efficiently targeted by the PD-1 antibody than CD8+ T cells. Both tumor-associated macrophages (TAMs) of monocyte origin, which are colony-stimulating factor 1 receptor (CSF1R) dependent, and TAMs of alveolar origin, which are sensitive to cisplatin, contributed to establish a transforming growth factor-ß-rich tumor microenvironment that supported PD-1+ Tregs Dual TAM targeting with a combination of a CSF1R inhibitor and cisplatin abated Tregs, redirected the PD-1 antibody to CD8+ T cells, and improved the efficacy of antiangiogenic immunotherapy, achieving regression of most tumors.

Macrophage depletion induces edema through release of matrix-degrading proteases and proteoglycan deposition.

Colony-stimulating factor 1 receptor (CSF1R) blockade abates tumor-associated macrophage (TAM) infiltrates and provides marked clinical benefits in diffuse-type tenosynovial giant cell tumors. However, facial edema is a common adverse event associated with TAM elimination in patients. In this study, we examined molecular and cellular events associated with edema formation in mice and human patients with cancer treated with a CSF1R blocking antibody. Extended antibody treatment of mice caused marked body weight gain, an indicator of enhanced body fluid retention. This was associated with an increase of extracellular matrix-remodeling metalloproteinases (MMPs), namely MMP2 and MMP3, and enhanced deposition of hyaluronan (HA) and proteoglycans, leading to skin thickening. Discontinuation of anti-CSF1R treatment or blockade of MMP activity restored unaltered body weight and normal skin morphology in the mice. In patients, edema developed at doses well below the established optimal biological dose for emactuzumab, a CSF1R dimerization inhibitor. Patients who developed edema in response to emactuzumab had elevated HA in peripheral blood. Our findings indicate that an early increase of peripheral HA can serve as a pharmacodynamic marker for edema development and suggest potential interventions based on MMP inhibition for relieving periorbital edema in patients treated with CSF1R inhibitors.

Imaging-based spectrometer-less optofluidic biosensors based on dielectric metasurfaces for detecting extracellular vesicles.

Biosensors are indispensable tools for public, global, and personalized healthcare as they provide tests that can be used from early disease detection and treatment monitoring to preventing pandemics. We introduce single-wavelength imaging biosensors capable of reconstructing spectral shift information induced by biomarkers dynamically using an advanced data processing technique based on an optimal linear estimator. Our method achieves superior sensitivity without wavelength scanning or spectroscopy instruments. We engineered diatomic dielectric metasurfaces supporting bound states in the continuum that allows high-quality resonances with accessible near-fields by in-plane symmetry breaking. The large-area metasurface chips are configured as microarrays and integrated with microfluidics on an imaging platform for real-time detection of breast cancer extracellular vesicles encompassing exosomes. The optofluidic system has high sensing performance with nearly 70 1/RIU figure-of-merit enabling detection of on average 0.41 nanoparticle/µm2 and real-time measurements of extracellular vesicles binding from down to 204 femtomolar solutions. Our biosensors provide the robustness of spectrometric approaches while substituting complex instrumentation with a single-wavelength light source and a complementary-metal-oxide-semiconductor camera, paving the way toward miniaturized devices for point-of-care diagnostics.

Disentangling the complexity of tumor-derived extracellular vesicles.

The tumor microenvironment encompasses an intertwined ensemble of both transformed cancer cells and non-transformed host cells, which together establish a signaling network that regulates tumor progression. By conveying both homo- and heterotypic cell-to-cell communication cues, tumor-derived extracellular vesicles (tEVs) modulate several cancer-associated processes, such as immunosuppression, angiogenesis, invasion, and metastasis. Herein we discuss how recent methodological advances in the isolation and characterization of tEVs may help to broaden our understanding of their functions in tumor biology and, potentially, establish their utility as cancer biomarkers.

Targeting LRG1 boosts immunotherapy.

Vascular normalization therapy has the potential to facilitate drug delivery and lymphocyte infiltration in tumors. Yet, optimal targets and dosage regimens remain elusive. In this issue of Med, O'Connor et al. show that inhibition of LRG1 stabilizes the tumor-associated vasculature to enhance tumor response to both cytotoxic and immune therapies.

Biology and therapeutic targeting of tumour-associated macrophages.

Macrophages sustain tumour progression by facilitating angiogenesis, promoting immunosuppression, and enhancing cancer cell invasion and metastasis. They also modulate tumour response to anti-cancer therapy in pre-clinical models. This knowledge has motivated the development of agents that target tumour-associated macrophages (TAMs), some of which have been investigated in early clinical trials. Here, we provide a comprehensive overview of the biology and therapeutic targeting of TAMs, highlighting opportunities, setbacks, and new challenges that have emerged after a decade of intense translational and clinical research into these multifaceted immune cells. © 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Antiangiogenic immunotherapy suppresses desmoplastic and chemoresistant intestinal tumors in mice.

Mutations in APC promote colorectal cancer (CRC) progression through uncontrolled WNT signaling. Patients with desmoplastic CRC have a significantly worse prognosis and do not benefit from chemotherapy, but the mechanisms underlying the differential responses of APC-mutant CRCs to chemotherapy are not well understood. We report that expression of the transcription factor prospero homeobox 1 (PROX1) was reduced in desmoplastic APC-mutant human CRCs. In genetic Apc-mutant mouse models, loss of Prox1 promoted the growth of desmoplastic, angiogenic, and immunologically silent tumors through derepression of Mmp14. Although chemotherapy inhibited Prox1-proficient tumors, it promoted further stromal activation, angiogenesis, and invasion in Prox1-deficient tumors. Blockade of vascular endothelial growth factor A (VEGFA) and angiopoietin-2 (ANGPT2) combined with CD40 agonistic antibodies promoted antiangiogenic and immunostimulatory reprogramming of Prox1-deficient tumors, destroyed tumor fibrosis, and unleashed T cell-mediated killing of cancer cells. These results pinpoint the mechanistic basis of chemotherapy-induced hyperprogression and illustrate a therapeutic strategy for chemoresistant and desmoplastic CRCs.

A LIGHTning Strike to the Metastatic Niche.

Tumor-induced vascular alterations in distant organs have been linked to the spreading of cancer. In this issue of Cell Reports, He et al. (2019) show that targeting the cytokine LIGHT to the pulmonary vasculature prevents the establishment of lung metastasis in mice.