Abstract
Peritoneal dialysis (PD) patients are at high risk for peritonitis, an infection of the peritoneum that affects 13% of PD users annually. Relying on subjective peritonitis symptoms results ...in delayed treatment, leading to high hospitalisation costs, peritoneal scarring, and premature transition to haemodialysis. We have developed and tested a low-cost, easy-to-use technology that uses microscopy and image analysis to screen for peritonitis across the effluent drain tube. Compared to other technologies, our prototype is made from off-the-shelf, low-cost materials. It can be set up quickly and key stakeholders believe it can improve the overall PD experience. We demonstrate that our prototype classifies infection-indicating and healthy white blood cell levels in clinically collected patient effluent with 94% accuracy. Integration of our technology into PD setups as a screening tool for peritonitis would enable earlier physician notification, allowing for prompt diagnosis and treatment to prevent hospitalisations, reduce scarring, and increase PD longevity. Our findings demonstrate the versatility of microscopy and image analysis for infection screening and are a proof of principle for their future applications in health care.
While CRISPR screens are helping uncover genes regulating many cell-intrinsic processes, existing approaches are suboptimal for identifying extracellular gene functions, particularly in the tissue ...context. Here, we developed an approach for spatial functional genomics called Perturb-map. We applied Perturb-map to knock out dozens of genes in parallel in a mouse model of lung cancer and simultaneously assessed how each knockout influenced tumor growth, histopathology, and immune composition. Moreover, we paired Perturb-map and spatial transcriptomics for unbiased analysis of CRISPR-edited tumors. We found that in Tgfbr2 knockout tumors, the tumor microenvironment (TME) was converted to a fibro-mucinous state, and T cells excluded, concomitant with upregulated TGFβ and TGFβ-mediated fibroblast activation, indicating that TGFβ-receptor loss on cancer cells increased TGFβ bioavailability and its immunosuppressive effects on the TME. These studies establish Perturb-map for functional genomics within the tissue at single-cell resolution with spatial architecture preserved and provide insight into how TGFβ responsiveness of cancer cells can affect the TME.
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•Multimodal phenotyping of CRISPR screens in situ by imaging and spatial transcriptomics•Imaging >100 cancer clones in tissue reveals the spatial organization of tumor clonality•Tgfbr2 KO on lung cancer cells promotes TME remodeling and immune exclusion•Loss of cancer cell TGFβ receptor leads to enhanced TGFβ activation of tumor stroma
A spatial functional genomics platform termed Perturb-map combines CRISPR pools, multiplex imaging, and spatial transcriptomics to enable the identification of genetic determinants of tumor composition, organization, and immunity.
Despite no apparent defects in T cell priming and recruitment to tumors, a large subset of T cell rich tumors fail to respond to immune checkpoint blockade (ICB). We leveraged a neoadjuvant anti-PD-1 ...trial in patients with hepatocellular carcinoma (HCC), as well as additional samples collected from patients treated off-label, to explore correlates of response to ICB within T cell-rich tumors. We show that ICB response correlated with the clonal expansion of intratumoral CXCL13
CH25H
IL-21
PD-1
CD4
T helper cells ("CXCL13
T
") and Granzyme K
PD-1
effector-like CD8
T cells, whereas terminally exhausted CD39
TOX
PD-1
CD8
T cells dominated in nonresponders. CD4
and CD8
T cell clones that expanded post-treatment were found in pretreatment biopsies. Notably, PD-1
TCF-1
(Progenitor-exhausted) CD8
T cells shared clones mainly with effector-like cells in responders or terminally exhausted cells in nonresponders, suggesting that local CD8
T cell differentiation occurs upon ICB. We found that these Progenitor CD8
T cells interact with CXCL13
T
within cellular triads around dendritic cells enriched in maturation and regulatory molecules, or "mregDC". These results suggest that discrete intratumoral niches that include mregDC and CXCL13
T
control the differentiation of tumor-specific Progenitor exhasuted CD8
T cells following ICB.
Prostate cancer is responsible for over 30,000 US deaths annually, attributed largely to incurable metastatic disease. Here, we demonstrate that high levels of plectin are associated with localized ...and metastatic human prostate cancer when compared to benign prostate tissues. Knock-down of plectin inhibits prostate cancer cell growth and colony formation in vitro, and growth of prostate cancer xenografts in vivo. Plectin knock-down further impairs aggressive and invasive cellular behavior assessed by migration, invasion, and wound healing in vitro. Consistently, plectin knock-down cells have impaired metastatic colonization to distant sites including liver, lung, kidney, bone, and genitourinary system. Plectin knock-down inhibited number of metastases per organ, as well as decreased overall metastatic burden. To gain insights into the role of plectin in prostate cancer growth and metastasis, we performed proteomic analysis of prostate cancer plectin knock-down xenograft tissues. Gene set enrichment analysis shows an increase in levels of proteins involved with extracellular matrix and laminin interactions, and a decrease in levels of proteins regulating amino acid metabolism, cytoskeletal proteins, and cellular response to stress. Collectively these findings demonstrate that plectin is an important regulator of prostate cancer cell growth and metastasis.
Despite their key regulatory role and therapeutic potency, the molecular signatures of interactions between T cells and antigen-presenting myeloid cells within the tumor microenvironment remain ...poorly characterized. Here, we systematically characterize these interactions using RNA sequencing of physically interacting cells (PIC-seq) and find that CD4
PD-1
CXCL13
T cells are a major interacting hub with antigen-presenting cells in the tumor microenvironment of human non-small cell lung carcinoma. We define this clonally expanded, tumor-specific and conserved T-cell subset as T-helper tumor (Tht) cells. Reconstitution of Tht cells in vitro and in an ovalbumin-specific αβ TCR CD4
T-cell mouse model, shows that the Tht program is primed in tumor-draining lymph nodes by dendritic cells presenting tumor antigens, and that their function is important for harnessing the antitumor response of anti-PD-1 treatment. Our molecular and functional findings support the modulation of Tht-dendritic cell interaction checkpoints as a major interventional strategy in immunotherapy.
Myeloid cells are known to suppress antitumour immunity
. However, the molecular drivers of immunosuppressive myeloid cell states are not well defined. Here we used single-cell RNA sequencing of ...human and mouse non-small cell lung cancer (NSCLC) lesions, and found that in both species the type 2 cytokine interleukin-4 (IL-4) was predicted to be the primary driver of the tumour-infiltrating monocyte-derived macrophage phenotype. Using a panel of conditional knockout mice, we found that only deletion of the IL-4 receptor IL-4Rα in early myeloid progenitors in bone marrow reduced tumour burden, whereas deletion of IL-4Rα in downstream mature myeloid cells had no effect. Mechanistically, IL-4 derived from bone marrow basophils and eosinophils acted on granulocyte-monocyte progenitors to transcriptionally programme the development of immunosuppressive tumour-promoting myeloid cells. Consequentially, depletion of basophils profoundly reduced tumour burden and normalized myelopoiesis. We subsequently initiated a clinical trial of the IL-4Rα blocking antibody dupilumab
given in conjunction with PD-1/PD-L1 checkpoint blockade in patients with relapsed or refractory NSCLC who had progressed on PD-1/PD-L1 blockade alone (ClinicalTrials.gov identifier NCT05013450 ). Dupilumab supplementation reduced circulating monocytes, expanded tumour-infiltrating CD8 T cells, and in one out of six patients, drove a near-complete clinical response two months after treatment. Our study defines a central role for IL-4 in controlling immunosuppressive myelopoiesis in cancer, identifies a novel combination therapy for immune checkpoint blockade in humans, and highlights cancer as a systemic malady that requires therapeutic strategies beyond the primary disease site.
Although it has been more than 2 years since the start of the coronavirus disease 2019 (COVID-19) pandemic, COVID-19 continues to be a worldwide health crisis. Despite the development of preventive ...vaccines, therapies to treat COVID-19 and other inflammatory diseases remain a major unmet need in medicine. Our study sought to identify drivers of disease severity and mortality to develop tailored immunotherapy strategies to halt disease progression. We assembled the Mount Sinai COVID-19 Biobank, which was composed of almost 600 hospitalized patients followed longitudinally through the peak of the pandemic in 2020. Moderate disease and survival were associated with a stronger antigen presentation and effector T cell signature. In contrast, severe disease and death were associated with an altered antigen presentation signature, increased numbers of inflammatory immature myeloid cells, and extrafollicular activated B cells that have been previously associated with autoantibody formation. In severely ill patients with COVID-19, lung tissue-resident alveolar macrophages not only were drastically depleted but also had an altered antigen presentation signature, which coincided with an influx of inflammatory monocytes and monocyte-derived macrophages. In addition, we found that the size of the alveolar macrophage pool correlated with patient outcome and that alveolar macrophage numbers and functionality were restored to homeostasis in patients who recovered from COVID-19. These data suggest that local and systemic myeloid cell dysregulation are drivers of COVID-19 severity and modulation of alveolar macrophage numbers and activity in the lung may be a viable therapeutic strategy for the treatment of critical inflammatory lung diseases.
Surgical resection of early stage hepatocellular carcinoma is standard clinical practice; however, most tumours recur despite surgery, and no perioperative intervention has shown a survival benefit. ...Neoadjuvant immunotherapy has induced pathological responses in multiple tumour types and might decrease the risk of postoperative recurrence in hepatocellular carcinoma. We aimed to evaluate the clinical activity of neoadjuvant cemiplimab (an anti-PD-1) in patients with resectable hepatocellular carcinoma.
For this single-arm, open-label, phase 2 trial, patients with resectable hepatocellular carcinoma (stage Ib, II, and IIIb) were enrolled and received two cycles of neoadjuvant cemiplimab 350 mg intravenously every 3 weeks followed by surgical resection. Eligible patients were aged 18 years or older, had confirmed resectable hepatocellular carcinoma, an Eastern Cooperative Oncology Group performance status of 0 or 1, and adequate liver function. Patients were excluded if they had metastatic disease, if the surgery was not expected to be curative, if they had a known additional malignancy requiring active treatment, or if they required systemic steroid treatment or any other immunosuppressive therapy. After resection, patients received an additional eight cycles of cemiplimab 350 mg intravenously every 3 weeks in the adjuvant setting. The primary endpoint was significant tumour necrosis on pathological examination (defined as >70% necrosis of the resected tumour). Secondary endpoints included delay of surgery, the proportion of patients with an overall response, change in CD8
T-cell density, and adverse events. Tumour necrosis and response were analysed in all patients who received at least one dose of cemiplimab and completed surgical resection; safety and other endpoints were analysed in the intention-to-treat population. Patients underwent pre-treatment biopsies and blood collection throughout treatment. This trial is registered with ClinicalTrials.gov (NCT03916627, Cohort B) and is ongoing.
Between Aug 5, 2019, and Nov 25, 2020, 21 patients were enrolled. All patients received neoadjuvant cemiplimab, and 20 patients underwent successful resection. Of the 20 patients with resected tumours, four (20%) had significant tumour necrosis. Three (15%) of 20 patients had a partial response, and all other patients maintained stable disease. 20 (95%) patients had a treatment-emergent adverse event of any grade during the neoadjuvant treatment period. The most common adverse events of any grade were increased aspartate aminotransferase (in four patients), increased blood creatine phosphokinase (in three), constipation (in three), and fatigue (in three). Seven patients had grade 3 adverse events, including increased blood creatine phosphokinase (in two patients) and hypoalbuminaemia (in one). No grade 4 or 5 events were observed. One patient developed pneumonitis, which led to a delay in surgery by 2 weeks.
This report is, to our knowledge, the largest clinical trial of a neoadjuvant anti-PD-1 monotherapy reported to date in hepatocellular carcinoma. The observed pathological responses to cemiplimab in this cohort support the design of larger trials to identify the optimal treatment duration and definitively establish the clinical benefit of preoperative PD-1 blockade in patients with hepatocellular carcinoma.
Regeneron Pharmaceuticals.
Among men, prostate cancer is the second leading cause of cancer-associated mortality, with advanced disease remaining a major clinical challenge. We describe a small molecule, SU086, as a ...therapeutic strategy for advanced prostate cancer. We demonstrate that SU086 inhibits the growth of prostate cancer cells in vitro, cell-line and patient-derived xenografts in vivo, and ex vivo prostate cancer patient specimens. Furthermore, SU086 in combination with standard of care second-generation anti-androgen therapies displays increased impairment of prostate cancer cell and tumor growth in vitro and in vivo. Cellular thermal shift assay reveals that SU086 binds to heat shock protein 90 (HSP90) and leads to a decrease in HSP90 levels. Proteomic profiling demonstrates that SU086 binds to and decreases HSP90. Metabolomic profiling reveals that SU086 leads to perturbation of glycolysis. Our study identifies SU086 as a treatment for advanced prostate cancer as a single agent or when combined with second-generation anti-androgens.
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•SU086 inhibits prostate cancer growth in preclinical models of prostate cancer•SU086 targets heat shock protein 90•SU086 alters prostate cancer glycolysis and decreases intratumoral metabolism•SU086 in combination with anti-androgens halts prostate cancer growth
Rice et al. identify SU086 as a therapeutic strategy for treatment of prostate cancer, as it impairs prostate cancer growth, inhibits HSP90, and impairs glycolysis and intratumoral metabolism. SU086 alone and in combination therapy strategies has strong therapeutic potential in pre-clinical models of prostate cancer.
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