A case for the study of native extracellular vesicles.

Three main areas of research revolve around extracellular vesicles (EVs): their use as early detection diagnostics for cancer prevention, engineering of EVs or other enveloped viral-like particles for therapeutic purposes and to understand how EVs impact biological processes. When investigating the biology of EVs, it is important to consider strategies able to track and alter EVs directly in vivo, as they are released by donor cells. This can be achieved by suitable engineering of EV donor cells, either before implantation or directly in vivo. Here, we make a case for the study of native EVs, that is, EVs released by cells living within a tissue. Novel genetic approaches to detect intercellular communications mediated by native EVs and profile recipient cells are discussed. The use of Rab35 dominant negative mutant is proposed for functional in vivo studies on the roles of native EVs. Ultimately, investigations on native EVs will tremendously advance our understanding of EV biology and open novel opportunities for therapy and prevention.

A drug-like molecule engages nuclear hormone receptor DAF-12/FXR to regulate mitophagy and extend lifespan.

Autophagy-lysosomal function is crucial for maintaining healthy lifespan and preventing age-related diseases. The transcription factor TFEB plays a key role in regulating this pathway. Decreased TFEB expression is associated with various age-related disorders, making it a promising therapeutic target. In this study, we screened a natural product library and discovered mitophagy-inducing coumarin (MIC), a benzocoumarin compound that enhances TFEB expression and lysosomal function. MIC robustly increases the lifespan of Caenorhabditis elegans in an HLH-30/TFEB-dependent and mitophagy-dependent manner involving DCT-1/BNIP3 while also preventing mitochondrial dysfunction in mammalian cells. Mechanistically, MIC acts by inhibiting ligand-induced activation of the nuclear hormone receptor DAF-12/FXR, which, in turn, induces mitophagy and extends lifespan. In conclusion, our study uncovers MIC as a promising drug-like molecule that enhances mitochondrial function and extends lifespan by targeting DAF-12/FXR. Furthermore, we discovered DAF-12/FXR as a previously unknown upstream regulator of HLH-30/TFEB and mitophagy.

Improving radiotherapy in immunosuppressive microenvironments by targeting complement receptor C5aR1.

An immunosuppressive microenvironment causes poor tumor T cell infiltration and is associated with reduced patient overall survival in colorectal cancer. How to improve treatment responses in these tumors is still a challenge. Using an integrated screening approach to identify cancer-specific vulnerabilities, we identified complement receptor C5aR1 as a druggable target, which when inhibited improved radiotherapy, even in tumors displaying immunosuppressive features and poor CD8+ T cell infiltration. While C5aR1 is well-known for its role in the immune compartment, we found that C5aR1 is also robustly expressed on malignant epithelial cells, highlighting potential tumor cell-specific functions. C5aR1 targeting resulted in increased NF-κB-dependent apoptosis specifically in tumors and not normal tissues, indicating that, in malignant cells, C5aR1 primarily regulated cell fate. Collectively, these data revealed that increased complement gene expression is part of the stress response mounted by irradiated tumors and that targeting C5aR1 could improve radiotherapy, even in tumors displaying immunosuppressive features.

Clinical and research updates on the VISTA immune checkpoint: immuno-oncology themes and highlights.

Immune checkpoints limit the activation of the immune system and serve an important homeostatic function but can also restrict immune responses against tumors. Inhibition of specific immune checkpoint proteins such as the B7:CD28 family members programmed cell death protein-1 (PD-1) and cytotoxic T-lymphocyte antigen-4 (CTLA-4) has transformed the treatment of various cancers by promoting the anti-tumor activation of immune cells. In contrast to these effects, the V-domain immunoglobulin suppressor of T-cell activation (VISTA) regulates the steady state of the resting immune system and promotes homeostasis by mechanisms distinct from PD-1 and CTLA-4. The effects of VISTA blockade have been shown to include a decrease in myeloid suppression coupled with proinflammatory changes by mechanisms that are separate and distinct from other immune checkpoint proteins; in some preclinical studies these immune effects appear synergistic. Given the potential benefits of VISTA blockade in the context of cancer therapy, the second Annual VISTA Symposium was convened virtually on September 23, 2022, to review new research from investigators and immuno-oncology experts. Discussions in the meeting extended the knowledge of VISTA biology and the effects of VISTA inhibition, particularly on cells of the myeloid lineage and resting T cells, as three candidate anti-VISTA antibodies are in, or nearing, clinical development.

Targeting DNA repair for cancer treatment: Lessons from PARP inhibitor trials.

Ionizing radiation is frequently used to treat solid tumors, as it causes DNA damage and kill cancer cells. However, damaged DNA is repaired involving poly-(ADP-ribose) polymerase-1 (PARP-1) causing resistance to radiation therapy. Thus, PARP-1 represents an important target in multiple cancer types, including prostate cancer. PARP is a nuclear enzyme essential for single-strand DNA breaks repair. Inhibiting PARP-1 is lethal in a wide range of cancer cells that lack the homologous recombination repair (HR) pathway. This article provides a concise and simplified overview of the development of PARP inhibitors in the laboratory and their clinical applications. We focused on the use of PARP inhibitors in various cancers, including prostate cancer. We also discussed some of the underlying principles and challenges that may affect the clinical efficacy of PARP inhibitors.

Galectin-1 Mediates Chronic STING Activation in Tumors to Promote Metastasis through MDSC Recruitment.

The immune system plays a crucial role in the regulation of metastasis. Tumor cells systemically change immune functions to facilitate metastatic progression. Through this study, we deciphered how tumoral galectin-1 (Gal1) expression shapes the systemic immune environment to promote metastasis in head and neck cancer (HNC). In multiple preclinical models of HNC and lung cancer in immunogenic mice, Gal1 fostered the establishment of a premetastatic niche through polymorphonuclear myeloid-derived suppressor cells (PMN-MDSC), which altered the local microenvironment to support metastatic spread. RNA sequencing of MDSCs from premetastatic lungs in these models demonstrated the role of PMN-MDSCs in collagen and extracellular matrix remodeling in the premetastatic compartment. Gal1 promoted MDSC accumulation in the premetastatic niche through the NF-κB signaling axis, triggering enhanced CXCL2-mediated MDSC migration. Mechanistically, Gal1 sustained NF-κB activation in tumor cells by enhancing stimulator of interferon gene (STING) protein stability, leading to prolonged inflammation-driven MDSC expansion. These findings suggest an unexpected protumoral role of STING activation in metastatic progression and establish Gal1 as an endogenous-positive regulator of STING in advanced-stage cancers. SIGNIFICANCE: Galectin-1 increases STING stability in cancer cells that activates NF-κB signaling and CXCL2 expression to promote MDSC trafficking, which stimulates the generation of a premetastatic niche and facilitates metastatic progression.

Decursin inhibits EGFR-ERK1/2 signaling axis in advanced human prostate carcinoma cells.

We have shown that decursin, a coumarin compound, induces cell cycle arrest and apoptosis in human prostate cancer cells (PCa); however, its molecular mechanisms are largely unexplored. We studied the mechanisms associated with its anticancer activity in advanced human prostate carcinoma cells. We found that decursin inhibited epidermal growth factor receptor (EGFR) signaling by inhibiting its activating phosphorylation at tyrosine 1068 residue in DU145 and 22Rv1 cells. This inhibition of EGFR was associated with the downregulation of ERK1/2 phosphorylation. Both EGFR and ERK1/2 are known to be deregulated/activated in many human malignancies. Consistent with our earlier study, decursin (25-100 µM) treatment for 24-72 h inhibited DU145 cell proliferation by 49%-87% (p < 0.001) which was associated with strong G1 phase arrest and cell death. It also decreased (p < 0.001) the number of surviving colonies. Decursin moderately increased the expression of Rb-related proteins p107 and p130 but decreased the levels of E2F family transcription factors including E2F-3, E2F-4 and E2F-5. Further, decursin strongly inhibited the growth of androgen-dependent prostate carcinoma 22Rv1 cells from 61% to 79% (p < 0.001) and arrested these cells at G1 phase via induction of cyclin-dependent kinase inhibitor p27/Kip1 and downregulation of CDK2 and CDK4 protein expression. Additionally, EGFR inhibitor erlotinib- and EGF ligand-modulated EGFR activation validated EGFR signaling as a target of decursin-mediated cell growth inhibition and cytotoxicity. Decursin decreased EGF ligand-induced phosphorylation of EGFR (Y-1068) as well as activation of its downstream mediator, ERK1/2. Furthermore, inhibitory targeting of EGFR-ERK1/2 axis by combinatorial treatment of decursin and erlotinib further sensitized DU145 cells for the decursin-induced growth inhibition and cell death. Overall, these findings strongly suggest that anticancer efficacy of decursin against human PCa involves inhibitory targeting of EGFR-ERK1/2 signaling axis, a pathway constitutively active in advanced PCa.

NFE2L2 Mutations Enhance Radioresistance in Head and Neck Cancer by Modulating Intratumoral Myeloid Cells.

Radiotherapy (RT) is one of the primary treatments of head and neck squamous cell carcinoma (HNSCC), which has a high-risk of locoregional failure (LRF). Presently, there is no reliable predictive biomarker of radioresistance in HNSCC. Here, we found that mutations in NFE2L2, which encodes Nrf2, are associated with a significantly higher rate of LRF in patients with oral cavity cancer treated with surgery and adjuvant (chemo)radiotherapy but not in those treated with surgery alone. Somatic mutation of NFE2L2 led to Nrf2 activation and radioresistance in HNSCC cells. Tumors harboring mutant Nrf2E79Q were substantially more radioresistant than tumors with wild-type Nrf2 in immunocompetent mice, whereas the difference was diminished in immunocompromised mice. Nrf2E79Q enhanced radioresistance through increased recruitment of intratumoral polymorphonuclear myeloid-derived suppressor cells (PMN-MDSC) and reduction of M1-polarized macrophages. Treatment with the glutaminase inhibitor CB-839 overcame the radioresistance induced by Nrf2E79Q or Nrf2E79K. RT increased expression of PMN-MDSC-attracting chemokines, including CXCL1, CXLC3, and CSF3, in Nrf2E79Q-expressing tumors via the TLR4, which could be reversed by CB-839. This study provides insights into the impact of NFE2L2 mutations on radioresistance and suggests that CB-839 can increase radiosensitivity by switching intratumoral myeloid cells to an antitumor phenotype, supporting clinical testing of CB-839 with RT in HNSCC with NFE2L2 mutations. SIGNIFICANCE: NFE2L2 mutations are predictive biomarkers of radioresistance in head and neck cancer and confer sensitivity to glutaminase inhibitors to overcome radioresistance.

The Combination of Radiotherapy and Complement C3a Inhibition Potentiates Natural Killer cell Functions Against Pancreatic Cancer.

Pancreatic cancer is one of the deadliest cancers, against which current immunotherapy strategies are not effective. Herein, we analyzed the immune cell composition of the tumor microenvironment of pancreatic cancer samples in The Cancer Genome Atlas and found that the presence of intratumoral NK cells correlates with survival. Subsequent analysis also indicated that NK cell exclusion from the microenvironment is found in a high percentage of clinical pancreatic cancers and in preclinical models of pancreatic cancer. Mechanistically, NK cell exclusion is regulated in part by complement C3a and its receptor signaling. Inhibition of the C3a receptor enhances NK cell infiltration in syngeneic mouse models of pancreatic cancer resulting in tumor growth delay. However, tumor growth inhibition mediated by NK cells is not sufficient alone for complete tumor regression, but is potentiated when combined with radiation therapy. Our findings indicate that although C3a inhibition is a promising approach to enhance NK cell-based immunotherapy against pancreatic cancer, its combination with radiation therapy hold greater therapeutic benefit.

Aldehyde dehydrogenase 3A1 deficiency leads to mitochondrial dysfunction and impacts salivary gland stem cell phenotype.

Adult salivary stem/progenitor cells (SSPC) have an intrinsic property to self-renew in order to maintain tissue architecture and homeostasis. Adult salivary glands have been documented to harbor SSPC, which have been shown to play a vital role in the regeneration of the glandular structures postradiation damage. We have previously demonstrated that activation of aldehyde dehydrogenase 3A1 (ALDH3A1) after radiation reduced aldehyde accumulation in SSPC, leading to less apoptosis and improved salivary function. We subsequently found that sustained pharmacological ALDH3A1 activation is critical to enhance regeneration of murine submandibular gland after radiation damage. Further investigation shows that ALDH3A1 function is crucial for SSPC self-renewal and survival even in the absence of radiation stress. Salivary glands from Aldh3a1 -/- mice have fewer acinar structures than wildtype mice. ALDH3A1 deletion or pharmacological inhibition in SSPC leads to a decrease in mitochondrial DNA copy number, lower expression of mitochondrial specific genes and proteins, structural abnormalities, lower membrane potential, and reduced cellular respiration. Loss or inhibition of ALDH3A1 also elevates ROS levels, depletes glutathione pool, and accumulates ALDH3A1 substrate 4-hydroxynonenal (4-HNE, a lipid peroxidation product), leading to decreased survival of murine SSPC that can be rescued by treatment with 4-HNE specific carbonyl scavengers. Our data indicate that ALDH3A1 activity protects mitochondrial function and is important for the regeneration activity of SSPC. This knowledge will help to guide our translational strategy of applying ALDH3A1 activators in the clinic to prevent radiation-related hyposalivation in head and neck cancer patients.

Y box binding protein 1 inhibition as a targeted therapy for ovarian cancer.

Y box binding protein 1 (YB-1) is a multifunctional protein associated with tumor progression and the emergence of treatment resistance (TR). Here, we report an azopodophyllotoxin small molecule, SU056, that potently inhibits tumor growth and progression via YB-1 inhibition. This YB-1 inhibitor inhibits cell proliferation, resistance to apoptosis in ovarian cancer (OC) cells, and arrests in the G1 phase. Inhibitor treatment leads to enrichment of proteins associated with apoptosis and RNA degradation pathways while downregulating spliceosome pathway. In vivo, SU056 independently restrains OC progression and exerts a synergistic effect with paclitaxel to further reduce disease progression with no observable liver toxicity. Moreover, in vitro mechanistic studies showed delayed disease progression via inhibition of drug efflux and multidrug resistance 1, and significantly lower neurotoxicity as compared with etoposide. These data suggest that YB-1 inhibition may be an effective strategy to reduce OC progression, antagonize TR, and decrease patient mortality.

The prevalence of alcohol and other drugs in fatal road crashes in Victoria, Australia.

BACKGROUND: Driving under the influence of drugs, including alcohol, is a globally recognised risk factor for road traffic crashes. While the prevalence of alcohol and other drugs in fatal road crashes has been examined in other countries, recent data investigating drug driving in fatal Australian crashes are limited. This study aimed to examine how the presence of alcohol and other drugs in fatal road trauma in Victoria has changed over time in different road users. METHODS: A population-based review of road trauma deaths was performed over the period of 01 July 2006 to 30 June 2016 in Victoria, Australia, using data from the National Coronial Information System (NCIS) and the Victorian State Trauma Registry (VSTR). Drugs were grouped according to type and analysed accordingly. Poisson regression models were used to determine change in incidence rates over the study period. RESULTS: There were 2287 road traffic fatalities with complete toxicology data (97% of all road traffic fatalities). Alcohol (blood alcohol concentration, BAC) was the most commonly detected drug (>0.001 g/100 mL: 21.1%; >0.05 g/100 mL: 18.4%), followed by opioids (17.3%), THC (13.1%), antidepressants (9.7%), benzodiazepines (8.8%), amphetamine-type stimulants (7.1%), ketamine (3.4%), antipsychotics (0.9%) and cocaine (0.2%). Trends demonstrated changing use over time with specific drugs. Alcohol positive road fatalities declined 9% per year in passenger car/4WD drivers (IRR = 0.91, 95% CI: 0.88-0.95). The incidence of strong opioids (oxycodone, fentanyl, morphine, and methadone) increased 6% per year (IRR = 1.06; 95% CI: 1.02-1.10). Methylamphetamine was detected in 6.6% of cases and showed a yearly increase of 7% (IRR = 1.07; 95% CI: 1.01-1.13). The incidence of THC remained unchanged over the period, observed in 13.1% of cases. Stronger opioids were more commonly detected among pedal cyclists (19.0%) and pedestrians (20.9%) while THC was more commonly detected among motorcyclists (19.8%) and other light vehicle drivers (17.6%). CONCLUSIONS: A decline in the prevalence of alcohol in fatalities suggests that law enforcement and public health strategies in Australia to address road fatalities and drink-driving may have had a positive effect. However, increases were observed in the incidence of other potentially impairing drugs including opioids and amphetamines, specifically methylamphetamine, indicating a concerning trend in road safety in Victoria that warrants further monitoring.

Novel Aza-podophyllotoxin derivative induces oxidative phosphorylation and cell death via AMPK activation in triple-negative breast cancer.

BACKGROUND: To circumvent Warburg effect, several clinical trials for different cancers are utilising a combinatorial approach using metabolic reprogramming and chemotherapeutic agents including metformin. The majority of these metabolic interventions work via indirectly activating AMP-activated protein kinase (AMPK) to alter cellular metabolism in favour of oxidative phosphorylation over aerobic glycolysis. The effect of these drugs is dependent on glycaemic and insulin conditions.  Therefore, development of small molecules, which can activate AMPK, irrespective of the energy state, may be a better approach for triple-negative breast cancer (TNBC) treatment. METHODS: Therapeutic effect of SU212 on TNBC cells was examined using in vitro and in vivo models. RESULTS: We developed and characterised the efficacy of novel AMPK activator (SU212) that selectively induces oxidative phosphorylation and decreases glycolysis in TNBC cells, while not affecting these pathways in normal cells.   SU212 accomplished this metabolic reprogramming by activating AMPK independent of energy stress and irrespective of the glycaemic/insulin state. This leads to mitotic phase arrest and apoptosis in TNBC cells. In vivo, SU212 inhibits tumour growth, cancer progression and metastasis. CONCLUSIONS: SU212 directly activates AMPK in TNBC cells, but does not hamper glucose metabolism in normal cells. Our study provides compelling preclinical data for further development of SU212 for the treatment of TNBC.

Ambulance dispatch of older patients following primary and secondary telephone triage in metropolitan Melbourne, Australia: a retrospective cohort study.

BACKGROUND: Most calls to ambulance result in emergency ambulance dispatch (direct dispatch) following primary telephone triage. Ambulance Victoria uses clinician-led secondary telephone triage for patients identified as low-acuity during primary triage to refer them to alternative care pathways; however, some are returned for ambulance dispatch (secondary dispatch). Older adult patients are frequent users of ambulance services; however, little is known about the appropriateness of subsequent secondary dispatches. OBJECTIVES: To examine the appropriateness of secondary dispatch through a comparison of the characteristics and ambulance outcomes of older patients dispatched an emergency ambulance via direct or secondary dispatch. DESIGN: A retrospective cohort study of ambulance patient data between September 2009 and June 2012 was conducted. SETTING: The secondary telephone triage service operated in metropolitan Melbourne, Victoria, Australia during the study period. PARTICIPANTS: There were 90 086 patients included aged 65 years and over who had an emergency ambulance dispatch via direct or secondary dispatch with one of the five most common secondary dispatch paramedic diagnoses. MAIN OUTCOME MEASURES: Descriptive analyses compared characteristics, treatment and transportation rates between direct and secondary dispatch patients. RESULTS: The dispatch groups were similar in demographics, vital signs and hospital transportation rates. However, secondary dispatch patients were half as likely to be treated by paramedics (OR 0.51; CI 0.48 to 0.55; p<0.001). Increasing age was associated with decreasing treatment (p<0.005) and increasing transportation rates (p<0.005). CONCLUSION: Secondary triage could identify patients who would ultimately be transported to an emergency department. However, the lower paramedic treatment rates suggest many secondary dispatch patients may have been suitable for referral to alternative low-acuity transport or referral options.

Patient-specific record linkage between emergency department and hospital admission data for a cohort of people who inject drugs: methodological considerations for frequent presenters.

BACKGROUND: People who inject drugs (PWID) have been identified as frequent users of emergency department (ED) and hospital inpatient services. The specific challenges of record linkage in cohorts with numerous administrative health records occurring in close proximity are not well understood. Here, we present a method for patient-specific record linkage of ED and hospital admission data for a cohort of PWID. METHODS: Data from 688 PWID were linked to two state-wide administrative health databases identifying all ED visits and hospital admissions for the cohort between January 2008 and June 2013. We linked patient-specific ED and hospital admissions data, using administrative date-time timestamps and pre-specified linkage criteria, to identify hospital admissions stemming from ED presentations for a given individual. The ability of standalone databases to identify linked ED visits or hospital admissions was examined. RESULTS: There were 3459 ED visits and 1877 hospital admissions identified during the study period. Thirty-four percent of ED visits were linked to hospital admissions. Most links had hospital admission timestamps in-between or identical to their ED visit timestamps (n = 1035, 87%). Allowing 24-h between ED visits and hospital admissions captured more linked records, but increased manual inspection requirements. In linked records (n = 1190), the ED 'departure status' variable correctly reflected subsequent hospital admission in only 68% of cases. The hospital 'admission type' variable was non-specific in identifying if a preceding ED visit had occurred. CONCLUSIONS: Linking ED visits with subsequent hospital admissions in PWID requires access to date and time variables for accurate temporal sorting, especially for same-day presentations. Selecting time-windows to capture linked records requires discretion. Researchers risk under-ascertainment of hospital admissions if using ED data alone.

The role of Glial cell derived neurotrophic factor in head and neck cancer.

Glial cell-derived neurotrophic factor (GDNF) is reported to promote the survival of neurons and salivary gland regeneration after radiation damage. This study investigated the effect of GDNF on cell migration, growth, and response to radiation in preclinical models of head and neck squamous cell carcinoma (HNSCC) and correlated GDNF expression to treatment outcomes in HNSCC patients. Our ultimate goal is to determine whether systemic administration of GDNF at high dose is safe for the management of hyposalivation or xerostomia in HNSCC patients. Three HPV-positive and three HPV-negative cell lines were examined for cell migration, growth, and clonogenic survival in vitro and tumor growth assay in vivo. Immunohistochemical staining of GDNF, its receptors GFRα1 and its co-receptor RET was performed on two independent HNSCC tissue microarrays (TMA) and correlated to treatment outcomes. Results showed that GDNF only enhanced cell migration in two HPV-positive cells at supra-physiologic doses, but not in HPV-negative cells. GDNF did not increase cell survival in the tested cell lines post-irradiation. Likewise, GDNF treatment affected neither tumor growth in vitro nor response to radiation in xenografts in two HPV-positive and two HPV-negative HNSCC models. High stromal expression of GDNF protein was associated with worse overall survival in HPV-negative HNSCC on multivariate analysis in a combined cohort of patients from Stanford University (n = 82) and Washington University (n = 189); however, the association between GDNF gene expression and worse survival was not confirmed in a separate group of HPV-negative HNSCC patients identified from the Cancer Genome Atlas (TCGA) database. Based on these data, we do not believe that GNDF is a safe systemic treatment to prevent or treat xerostomia in HNSCC and a local delivery approach such as intraglandular injection needs to be explored.

Variations in the care of agitated patients in Australia and New Zealand ambulance services.

OBJECTIVE: The objective of the present study is to examine variations in paramedic care of the agitated patient, including verbal de-escalation, physical restraint and sedation, provided by ambulance services in Australia and New Zealand. METHODS: To examine the care of agitated patients, we first identified and reviewed all clinical practice guidelines for the management of agitated patients in Australian and New Zealand ambulance services between September and November 2018. We then conducted a structured questionnaire to obtain further information on the training, assessment and care of agitated patients by the ambulance services. Two authors extracted the data independently, and all interpretations and results were reviewed and confirmed by relevant ambulance services. RESULTS: There were 10 independent clinical practice guidelines for the care of agitated patients in the 10 ambulance services. All services reported training in the management of agitated patients, and two services used a validated tool to assess the level of agitation. All services used physical restraint, although six services required police presence to restrain the patient. All ambulance services used some form of sedation, typically divided into the management of mild to moderate, and severe agitation. The most common agent for sedation was midazolam, while ketamine was the most common agent for sedating severely agitated patients. The maximum dose was varied, and contraindications for sedating agents varied between services. CONCLUSIONS: There were wide variations across the ambulance services in terms of the assessment of agitation, as well as the use of physical restraint and sedation.

Galectin-1-driven T cell exclusion in the tumor endothelium promotes immunotherapy resistance.

Immune checkpoint inhibitors (ICIs), although promising, have variable benefit in head and neck cancer (HNC). We noted that tumor galectin-1 (Gal1) levels were inversely correlated with treatment response and survival in patients with HNC who were treated with ICIs. Using multiple HNC mouse models, we show that tumor-secreted Gal1 mediates immune evasion by preventing T cell migration into the tumor. Mechanistically, Gal1 reprograms the tumor endothelium to upregulate cell-surface programmed death ligand 1 (PD-L1) and galectin-9. Using genetic and pharmacological approaches, we show that Gal1 blockade increases intratumoral T cell infiltration, leading to a better response to anti-PD1 therapy with or without radiotherapy. Our study reveals the function of Gal1 in transforming the tumor endothelium into an immune-suppressive barrier and that its inhibition synergizes with ICIs.

The Immune Subtypes and Landscape of Squamous Cell Carcinoma.

PURPOSE: To identify immune subtypes and investigate the immune landscape of squamous cell carcinomas (SCC), which share common etiology and histologic features. EXPERIMENTAL DESIGN: Based on the immune gene expression profiles of 1,368 patients with SCC in the Cancer Genome Atlas (TCGA), we used consensus clustering to identify robust clusters of patients and assessed their reproducibility in an independent pan-SCC cohort of 938 patients. We further applied graph structure learning-based dimensionality reduction to the immune profiles to visualize the distribution of individual patients. RESULTS: We identified and independently validated six reproducible immune subtypes associated with distinct molecular characteristics and clinical outcomes. An immune-cold subtype had the least amount of lymphocyte infiltration and a high level of aneuploidy, and these patients had the worst prognosis. By contrast, an immune-hot subtype demonstrated the highest infiltration of CD8+ T cells, activated NK cells, and elevated IFNγ response. Accordingly, these patients had the best prognosis. A third subtype was dominated by M2-polarized macrophages with potent immune-suppressive factors such as TGFß signaling and reactive stroma, and these patients had relatively inferior prognosis. Other subtypes showed more diverse immunologic features with intermediate prognoses. Finally, our analysis revealed a complex immune landscape consisting of both discrete clusters and continuous spectrum. CONCLUSIONS: This study provides a conceptual framework to understand the tumor immune microenvironment of SCCs. Future work is needed to evaluate its relevance in the design of combination treatment strategies and guiding optimal selection of patients for immunotherapy.