Protocol to quantify immune cell distribution from the vasculature to the glioma microenvironment on sequential immunofluorescence multiplex images.

Although myeloid-derived immune cells can be dispersed throughout the tumor microenvironment (TME), anti-tumor effector cells are confined to the perivascular space. Here, we present a protocol to quantify immune cell distribution from tumor vasculature to its glioma microenvironment on sequential immunofluorescence multiplex images. We describe steps for sequential immunofluorescence multiplex staining, image generation, and storage. We then detail the procedures for tissue, vessel, and nuclei segmentation; cell phenotyping; data extraction; and training using RStudio and Spyder.

Targeting sinonasal undifferentiated carcinoma with a combinatory immunotherapy approach.

PURPOSE: Sinonasal undifferentiated carcinoma (SNUC) is a rare, aggressive malignancy of the sinonasal cavity with poor prognosis and limited treatment options. To investigate the potential for SNUC sensitivity to combinatory immunotherapy, we performed in vitro studies with SNUC cell lines and used multi-spectral immunofluorescence to characterize the in vivo patient SNUC tumor immune microenvironment (TIME). EXPERIMENTAL DESIGN: Human-derived SNUC cell lines were used for in vitro studies of tumor cell susceptibility to natural killer (NK) cell-based immunotherapeutic strategies. Tumor samples from 14 treatment naïve SNUC patients were examined via multi-spectral immunofluorescence and clinical correlations assessed. RESULTS: Anti-PD-L1 blockade enhanced NK cell lysis of SNUC cell lines ∼5.4 fold (P ≤ 0.0001). This effect was blocked by a CD16 neutralizing antibody demonstrating activity through an antibody-dependent cellular cytotoxicity (ADCC) mediated pathway. ADCC-dependent lysis of SNUC cells was further enhanced by upregulation of PD-L1 on tumor cells by exogenous interferon-gamma (IFN-γ) administration or interleukin-15 (IL-15) stimulated IFN-γ release from NK cells. Combination treatment with anti-PD-L1 blockade and IL-15 superagonism enhanced NK-cell killing of SNUC cells 9.6-fold (P ≤ 0.0001). Untreated SNUC patient tumor samples were found to have an NK cell infiltrate and PD-L1+ tumor cells at a median of 5.4 cells per mm2. A striking 55.7-fold increase in CKlow tumor cell/NK cell interactions was observed in patients without disease recurrence after treatment (P = 0.022). Patients with higher CD3+CD8+ in the stroma had a significantly improved 5-year overall survival (P = 0.0029) and a significant increase in CKlow tumor cell/CD8+ cytotoxic T cell interactions was noted in long-term survivors (P = 0.0225). CONCLUSION: These data provide the pre-clinical rationale for ongoing investigation into combinatory immunotherapy approaches for SNUC.

Molecular, Metabolic, and Subcellular Mapping of the Tumor Immune Microenvironment via 3D Targeted and Non-Targeted Multiplex Multi-Omics Analyses.

Most platforms used for the molecular reconstruction of the tumor-immune microenvironment (TIME) of a solid tumor fail to explore the spatial context of the three-dimensional (3D) space of the tumor at a single-cell resolution, and thus lack information about cell-cell or cell-extracellular matrix (ECM) interactions. To address this issue, a pipeline which integrated multiplex spatially resolved multi-omics platforms was developed to identify crosstalk signaling networks among various cell types and the ECM in the 3D TIME of two FFPE (formalin-fixed paraffin embedded) gynecologic tumor samples. These platforms include non-targeted mass spectrometry imaging (glycans, metabolites, and peptides) and Stereo-seq (spatial transcriptomics) and targeted seqIF (IHC proteomics). The spatially resolved imaging data in a two- and three-dimensional space demonstrated various cellular neighborhoods in both samples. The collection of spatially resolved analytes in a voxel (3D pixel) across serial sections of the tissue was also demonstrated. Data collected from this analytical pipeline were used to construct spatial 3D maps with single-cell resolution, which revealed cell identity, activation, and energized status. These maps will provide not only insights into the molecular basis of spatial cell heterogeneity in the TIME, but also novel predictive biomarkers and therapeutic targets, which can improve patient survival rates.

Comprehensive characterization of IFNγ signaling in acute myeloid leukemia reveals prognostic and therapeutic strategies.

Interferon gamma (IFNγ) is a critical cytokine known for its diverse roles in immune regulation, inflammation, and tumor surveillance. However, while IFNγ levels were elevated in sera of most newly diagnosed acute myeloid leukemia (AML) patients, its complex interplay in AML remains insufficiently understood. We aim to characterize these complex interactions through comprehensive bulk and single-cell approaches in bone marrow of newly diagnosed AML patients. We identify monocytic AML as having a unique microenvironment characterized by IFNγ producing T and NK cells, high IFNγ signaling, and immunosuppressive features. IFNγ signaling score strongly correlates with venetoclax resistance in primary AML patient cells. Additionally, IFNγ treatment of primary AML patient cells increased venetoclax resistance. Lastly, a parsimonious 47-gene IFNγ score demonstrates robust prognostic value. In summary, our findings suggest that inhibiting IFNγ is a potential treatment strategy to overcoming venetoclax resistance and immune evasion in AML patients.

Increased expression of CD70 in relapsed acute myeloid leukemia after hypomethylating agents.

Acute myeloid leukemia (AML) is the most common acute leukemia in adults. While induction chemotherapy leads to remission in most patients, a significant number will experience relapse. Therefore, there is a need for novel therapies that can improve remission rates in patients with relapsed and refractory AML. CD70 is the natural ligand for CD27 (a member of the TNF superfamily) and appears to be a promising therapeutic target. Consequently, there is considerable interest in developing chimeric antigen receptor (CAR) T-cell therapy products that can specifically target CD70 in various neoplasms, including AML. In this study, we employed routine diagnostic techniques, such as immunohistochemistry and flow cytometry, to investigate the expression of CD70 in bone marrow samples from treatment-naïve and relapsed AML patients after hypomethylating agents (HMA). Also, we evaluated the impact of HMA on CD70 expression and examined CD70 expression in various leukemic cell subsets and normal hematopoietic progenitors.

Mutant p53 gains oncogenic functions through a chromosomal instability-induced cytosolic DNA response.

Inactivating TP53 mutations leads to a loss of function of p53, but can also often result in oncogenic gain-of-function (GOF) of mutant p53 (mutp53) proteins which promotes tumor development and progression. The GOF activities of TP53 mutations are well documented, but the mechanisms involved remain poorly understood. Here, we study the mutp53 interactome and find that by targeting minichromosome maintenance complex components (MCMs), GOF mutp53 predisposes cells to replication stress and chromosomal instability (CIN), leading to a tumor cell-autonomous and cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING)-dependent cytosolic DNA response that activates downstream non-canonical nuclear factor kappa light chain enhancer of activated B cell (NC-NF-κB) signaling. Consequently, GOF mutp53-MCMs-CIN-cytosolic DNA-cGAS-STING-NC-NF-κB signaling promotes tumor cell metastasis and an immunosuppressive tumor microenvironment through antagonizing interferon signaling and regulating genes associated with pro-tumorigenic inflammation. Our findings have important implications for understanding not only the GOF activities of TP53 mutations but also the genome-guardian role of p53 and its inactivation during tumor development and progression.

Heterogeneity in intrahepatic macrophage populations and druggable target expression in patients with steatotic liver disease-related fibrosis.

Background & Aims: Clinical trials for reducing fibrosis in steatotic liver disease (SLD) have targeted macrophages with variable results. We evaluated intrahepatic macrophages in patients with SLD to determine if activity scores or fibrosis stages influenced phenotypes and expression of druggable targets, such as CCR2 and galectin-3. Methods: Liver biopsies from controls or patients with minimal or advanced fibrosis were subject to gene expression analysis using nCounter to determine differences in macrophage-related genes (n = 30). To investigate variability among individual patients, we compared additional biopsies by staining them with multiplex antibody panels (CD68/CD14/CD16/CD163/Mac387 or CD163/CCR2/galectin-3/Mac387) followed by spectral imaging and spatial analysis. Algorithms that utilize deep learning/artificial intelligence were applied to create cell cluster plots, phenotype profile maps, and to determine levels of protein expression (n = 34). Results: Several genes known to be pro-fibrotic (e.g. CD206, TREM2, CD163, and ARG1) showed either no significant differences or significantly decreased with advanced fibrosis. Although marked variability in gene expression was observed in individual patients with cirrhosis, several druggable targets and their ligands (e.g. CCR2, CCR5, CCL2, CCL5, and LGALS3) were significantly increased when compared to patients with minimal fibrosis. Antibody panels identified populations that were significantly increased (e.g. Mac387+), decreased (e.g. CD14+), or enriched (e.g. interactions of Mac387) in patients that had progression of disease or advanced fibrosis. Despite heterogeneity in patients with SLD, several macrophage phenotypes and druggable targets showed a positive correlation with increasing NAFLD activity scores and fibrosis stages. Conclusions: Patients with SLD have markedly varied macrophage- and druggable target-related gene and protein expression in their livers. Several patients had relatively high expression, while others were like controls. Overall, patients with more advanced disease had significantly higher expression of CCR2 and galectin-3 at both the gene and protein levels. Impact and implications: Appreciating individual differences within the hepatic microenvironment of patients with SLD may be paramount to developing effective treatments. These results may explain why such a small percentage of patients have responded to macrophage-targeting therapies and provide additional support for precision medicine-guided treatment of chronic liver diseases.

Cytosolic DNA accumulation promotes breast cancer immunogenicity via a STING-independent pathway.

BACKGROUND: Immune checkpoint blockade (ICB) has revolutionized cancer treatment. However, ICB alone has demonstrated only benefit in a small subset of patients with breast cancer. Recent studies have shown that agents targeting DNA damage response improve the efficacy of ICB and promote cytosolic DNA accumulation. However, recent clinical trials have shown that these agents are associated with hematological toxicities. More effective therapeutic strategies are urgently needed. METHODS: Primary triple negative breast cancer tumors were stained for cytosolic single-stranded DNA (ssDNA) using multiplex immunohistochemical staining. To increase cytosolic ssDNA, we genetically silenced TREX1. The role of tumor cytosolic ssDNA in promoting tumor immunogenicity and antitumor immune response was evaluated using murine breast cancer models. RESULTS: We found the tumorous cytosolic ssDNA is associated with tumor-infiltrating lymphocyte in patients with triple negative breast cancer. TREX1 deficiency triggered a STING-independent innate immune response via DDX3X. Cytosolic ssDNA accumulation in tumors due to TREX1 deletion is sufficient to drastically improve the efficacy of ICB. We further identified a cytosolic ssDNA inducer CEP-701, which sensitized breast tumors to ICB without the toxicities associated with inhibiting DNA damage response. CONCLUSIONS: This work demonstrated that cytosolic ssDNA accumulation promotes breast cancer immunogenicity and may be a novel therapeutic strategy to improve the efficacy of ICB with minimal toxicities.

Inhibition of Microsomal Prostaglandin E2 Synthase Reduces Collagen Deposition in Melanoma Tumors and May Improve Immunotherapy Efficacy by Reducing T-cell Exhaustion.

The arachidonic acid pathway participates in immunosuppression in various types of cancer. Our previous observation detailed that microsomal prostaglandin E2 synthase 1 (mPGES-1), an enzyme downstream of cyclooxygenase 2 (COX-2), limited antitumor immunity in melanoma; in addition, genetic depletion of mPGES-1 specifically enhanced immune checkpoint blockade therapy. The current study set out to distinguish the roles of mPGES-1 from those of COX-2 in tumor immunity and determine the potential of mPGES-1 inhibitors for reinforcing immunotherapy in melanoma. Genetic deletion of mPGES-1 showed different profiles of prostaglandin metabolites from that of COX-2 deletion. In our syngeneic mouse model, mPGES-1-deficient cells exhibited similar tumorigenicity to that of COX-2-deficient cells, despite a lower ability to suppress PGE2 synthesis by mPGES-1 depletion, indicating the presence of factors other than PGE2 that are likely to regulate tumor immunity. RNA-sequencing analysis revealed that mPGES-1 depletion reduced the expressions of collagen-related genes, which have been found to be associated with immunosuppressive signatures. In our mouse model, collagen was reduced in mPGES-1-deficient tumors, and phenotypic analysis of tumor-infiltrating lymphocytes indicated that mPGES-1-deficient tumors had fewer TIM3+ exhausted CD8+ T cells compared with COX-2-deficient tumors. CAY10678, an mPGES-1 inhibitor, was equivalent to celecoxib, a selective COX-2 inhibitor, in reinforcing anti-PD-1 treatment. Our study indicates that mPGES-1 inhibitors represent a promising adjuvant for immunotherapies in melanoma by reducing collagen deposition and T-cell exhaustion. Significance: Collagen is a predominant component of the extracellular matrix that may influence the tumor immune microenvironment for cancer progression. We present here that mPGES-1 has specific roles in regulating tumor immunity, associated with several collagen-related genes and propose that pharmacologic inhibition of mPGES-1 may hold therapeutic promise for improving immune checkpoint-based therapies.

Inflammation induced by tumor-associated nerves promotes resistance to anti-PD-1 therapy in cancer patients and is targetable by interleukin-6 blockade.

While the nervous system has reciprocal interactions with both cancer and the immune system, little is known about the potential role of tumor associated nerves (TANs) in modulating anti-tumoral immunity. Moreover, while peri-neural invasion is a well establish poor prognostic factor across cancer types, the mechanisms driving this clinical effect remain unknown. Here, we provide clinical and mechniastic association between TANs damage and resistance to anti-PD-1 therapy. Using electron microscopy, electrical conduction studies, and tumor samples of cutaneous squamous cell carcinoma (cSCC) patients, we showed that cancer cells can destroy myelin sheath and induce TANs degeneration. Multi-omics and spatial analyses of tumor samples from cSCC patients who underwent neoadjuvant anti-PD-1 therapy demonstrated that anti-PD-1 non-responders had higher rates of peri-neural invasion, TANs damage and degeneration compared to responders, both at baseline and following neoadjuvant treatment. Tumors from non-responders were also characterized by a sustained signaling of interferon type I (IFN-I) - known to both propagate nerve degeneration and to dampen anti-tumoral immunity. Peri-neural niches of non-responders were characterized by higher immune activity compared to responders, including immune-suppressive activity of M2 macrophages, and T regulatory cells. This tumor promoting inflammation expanded to the rest of the tumor microenvironment in non-responders. Anti-PD-1 efficacy was dampened by inducing nerve damage prior to treatment administration in a murine model. In contrast, anti-PD-1 efficacy was enhanced by denervation and by interleukin-6 blockade. These findings suggested a potential novel anti-PD-1 resistance drived by TANs damage and inflammation. This resistance mechanism is targetable and may have therapeutic implications in other neurotropic cancers with poor response to anti-PD-1 therapy such as pancreatic, prostate, and breast cancers.

Single-Cell Profiling of CD8+ T Cells in Acute Myeloid Leukemia Reveals a Continuous Spectrum of Differentiation and Clonal Hyperexpansion.

Comprehensive investigation of CD8+ T cells in acute myeloid leukemia (AML) is essential for developing immunotherapeutic strategies beyond immune checkpoint blockade. Herein, we performed single-cell RNA profiling of CD8+ T cells from 3 healthy bone marrow donors and 23 newly diagnosed (NewlyDx) and 8 relapsed/refractory (RelRef) patients with AML. Cells coexpressing canonical exhaustion markers formed a cluster constituting <1% of all CD8+ T cells. We identified two effector CD8+ T-cell subsets characterized by distinct cytokine and metabolic profiles that were differentially enriched in NewlyDx and RelRef patients. We refined a 25-gene CD8-derived signature correlating with therapy resistance, including genes associated with activation, chemoresistance, and terminal differentiation. Pseudotemporal trajectory analysis supported enrichment of a terminally differentiated state in CD8+ T cells with high CD8-derived signature expression at relapse or refractory disease. Higher expression of the 25-gene CD8 AML signature correlated with poorer outcomes in previously untreated patients with AML, suggesting that the bona fide state of CD8+ T cells and their degree of differentiation are clinically relevant. Immune clonotype tracking revealed more phenotypic transitions in CD8 clonotypes in NewlyDx than in RelRef patients. Furthermore, CD8+ T cells from RelRef patients had a higher degree of clonal hyperexpansion associated with terminal differentiation and higher CD8-derived signature expression. Clonotype-derived antigen prediction revealed that most previously unreported clonotypes were patient-specific, suggesting significant heterogeneity in AML immunogenicity. Thus, immunologic reconstitution in AML is likely to be most successful at earlier disease stages when CD8+ T cells are less differentiated and have greater capacity for clonotype transitions.

Spatial Transcriptomics of Intraductal Papillary Mucinous Neoplasms of the Pancreas Identifies NKX6-2 as a Driver of Gastric Differentiation and Indolent Biological Potential.

Intraductal papillary mucinous neoplasms (IPMN) of the pancreas are bona fide precursor lesions of pancreatic ductal adenocarcinoma (PDAC). The most common subtype of IPMNs harbors a gastric foveolar-type epithelium, and these low-grade mucinous neoplasms are harbingers of IPMNs with high-grade dysplasia and cancer. The molecular underpinning of gastric differentiation in IPMNs is unknown, although identifying drivers of this indolent phenotype might enable opportunities for intercepting progression to high-grade IPMN and cancer. We conducted spatial transcriptomics on a cohort of IPMNs, followed by orthogonal and cross-species validation studies, which established the transcription factor NKX6-2 as a key determinant of gastric cell identity in low-grade IPMNs. Loss of NKX6-2 expression is a consistent feature of IPMN progression, while reexpression of Nkx6-2 in murine IPMN lines recapitulates the aforementioned gastric transcriptional program and glandular morphology. Our study identifies NKX6-2 as a previously unknown transcription factor driving indolent gastric differentiation in IPMN pathogenesis. SIGNIFICANCE: Identification of the molecular features driving IPMN development and differentiation is critical to prevent cancer progression and enhance risk stratification. We used spatial profiling to characterize the epithelium and microenvironment of IPMN, which revealed a previously unknown link between NKX6-2 and gastric differentiation, the latter associated with indolent biological potential. See related commentary by Ben-Shmuel and Scherz-Shouval, p. 1768. This article is highlighted in the In This Issue feature, p. 1749.

An ERK5-NRF2 Axis Mediates Senescence-Associated Stemness and Atherosclerosis.

BACKGROUND: ERK5 (extracellular signal-regulated kinase 5) is a dual kinase transcription factor containing an N-terminal kinase domain and a C-terminal transcriptional activation domain. Many ERK5 kinase inhibitors have been developed and tested to treat cancer and inflammatory diseases. However, recent data have raised questions about the role of the catalytic activity of ERK5 in proliferation and inflammation. We aimed to investigate how ERK5 reprograms myeloid cells to the proinflammatory senescent phenotype, subsequently leading to atherosclerosis. METHODS: A ERK5 S496A (dephosphorylation mimic) knock in (KI) mouse model was generated using CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat-associated 9), and atherosclerosis was characterized by hypercholesterolemia induction. The plaque phenotyping in homozygous ERK5 S496A KI and wild type (WT) mice was studied using imaging mass cytometry. Bone marrow-derived macrophages were isolated from hypercholesterolemic mice and characterized using RNA sequencing and functional in vitro approaches, including senescence, mitochondria reactive oxygen species, and inflammation assays, as well as by metabolic extracellular flux analysis. RESULTS: We show that atherosclerosis was inhibited in ERK5 S496A KI mice. Furthermore, ERK5 S496 phosphorylation mediates both senescence-associated secretory phenotype and senescence-associated stemness by upregulating AHR (aryl hydrocarbon receptor) in plaque and bone marrow-derived macrophages isolated from hypercholesterolemic mice. We also discovered that ERK5 S496 phosphorylation could induce NRF2 (NFE2-related factor 2) SUMOylation at a novel K518 site to inhibit NRF2 transcriptional activity without altering ERK5 catalytic activity and mediates oxidized LDL (low-density lipoprotein)-induced senescence-associated secretory phenotype. Specific ERK5 kinase inhibitors (AX15836 and XMD8-92) also inhibited ERK5 S496 phosphorylation, suggesting the involvement of ERK5 S496 phosphorylation in the anti-inflammatory effects of these ERK5 kinase inhibitors. CONCLUSIONS: We discovered a novel mechanism by which the macrophage ERK5-NRF2 axis develops a unique senescence-associated secretory phenotype/stemness phenotype by upregulating AHR to engender atherogenesis. The finding of senescence-associated stemness phenotype provides a molecular explanation to resolve the paradox of senescence in proliferative plaque by permitting myeloid cells to escape the senescence-induced cell cycle arrest during atherosclerosis formation.

Mdm2/p53 levels in bone marrow mesenchymal stromal cells are essential for maintaining the hematopoietic niche in response to DNA damage.

Mesenchymal stromal cells (MSCs) are a key component of the bone marrow (BM) niche, providing essential support required for the maintenance of hematopoietic stem cells. To advance our understanding of physiological functions of p53 and Mdm2 in BM-MSCs, we developed traceable conditional mouse models targeting Mdm2 and/or Trp53 in vivo. We demonstrate that Mdm2 is essential for the emergence, maintenance, and hematopoietic support of BM-MSCs. Mdm2 haploinsufficiency in BM-MSCs resulted in genotoxic stress-associated thrombocytopenia, suggesting a functional role for Mdm2 in hematopoiesis. In a syngeneic mouse model of acute myeloid leukemia (AML), Trp53 deletion in BM-MSCs improved survival, and protected BM against hematopoietic toxicity from a murine Mdm2i, DS-5272. The transcriptional changes were associated with dysregulation of glycolysis, gluconeogenesis, and Hif-1α in BM-MSCs. Our results reveal a physiologic function of Mdm2 in BM-MSC, identify a previously unknown role of p53 pathway in BM-MSC-mediated support in AML and expand our understanding of the mechanism of hematopoietic toxicity of MDM2is.

Spatial Immunoprofiling of Adenoid Cystic Carcinoma Reveals B7-H4 Is a Therapeutic Target for Aggressive Tumors.

PURPOSE: Adenoid cystic carcinoma (ACC) is a heterogeneous malignancy, and no effective systemic therapy exists for metastatic disease. We previously described two prognostic ACC molecular subtypes with distinct therapeutic vulnerabilities, ACC-I and ACC-II. In this study, we explored the ACC tumor microenvironment (TME) using RNA-sequencing and spatial biology to identify potential therapeutic targets. EXPERIMENTAL DESIGN: Tumor samples from 62 ACC patients with available RNA-sequencing data that had been collected as part of previous studies were stained with a panel of 28 validated metal-tagged antibodies. Imaging mass cytometry (IMC) was performed using the Fluidigm Helios CyTOF instrument and analyzed with Visiopharm software. The B7-H4 antibody-drug conjugate AZD8205 was tested in ACC patient-derived xenografts (PDX). RESULTS: RNA deconvolution revealed that most ACCs are immunologically "cold," with approximately 30% being "hot." ACC-I tumors with a poor prognosis harbored a higher density of immune cells; however, spatial analysis by IMC revealed that ACC-I immune cells were significantly restricted to the stroma, characterizing an immune-excluded TME. ACC-I tumors overexpressed the immune checkpoint B7-H4, and the degree of immune exclusion was directly correlated with B7-H4 expression levels, an independent predictor of poor survival. Two ACC-I/B7-H4-high PDXs obtained 90% complete responses to a single dose of AZD8205, but none were observed with isotype-conjugated payload or in an ACC-II/B7-H4 low PDX. CONCLUSIONS: Spatial analysis revealed that ACC subtypes have distinct TMEs, with enrichment of ACC-I immune cells that are restricted to the stroma. B7-H4 is highly expressed in poor-prognosis ACC-I subtype and is a potential therapeutic target.

Single-Cell Profiling of CD8+ T Cells in Acute Myeloid Leukemia Reveals a Continuous Spectrum of Differentiation and Clonal Hyperexpansion.

Comprehensive investigation of CD8+ T cells in acute myeloid leukemia (AML) is essential for developing immunotherapeutic strategies beyond immune checkpoint blockade. Herein, we performed single-cell RNA profiling of CD8+ T cells from 3 healthy bone marrow donors and 23 newly diagnosed (NewlyDx) and 8 relapsed/refractory (RelRef) AML patients. Cells co-expressing canonical exhaustion markers formed a cluster constituting <1% of all CD8+ T cells. We identified two effector CD8+ T cell subsets characterized by distinct cytokine and metabolic profiles that were differentially enriched in NewlyDx and RelRef patients. We refined a 25-gene CD8-derived signature correlating with therapy resistance, including genes associated with activation, chemoresistance, and terminal differentiation. Pseudotemporal trajectory analysis supported enrichment of a terminally differentiated state in CD8+ T cells with high CD8-derived signature expression at relapse or refractory disease. Higher expression of the 25-gene CD8 AML signature correlated with poorer outcomes in previously untreated AML patients, suggesting that the bona fide state of CD8+ T cells and their degree of differentiation are clinically relevant. Immune clonotype tracking revealed more phenotypic transitions in CD8 clonotypes in NewlyDx than in RelRef patients. Furthermore, CD8+ T cells from RelRef patients had a higher degree of clonal hyperexpansion associated with terminal differentiation and higher CD8-derived signature expression. Clonotype-derived antigen prediction revealed that most previously unreported clonotypes were patient-specific, suggesting significant heterogeneity in AML immunogenicity. Thus, immunologic reconstitution in AML is likely to be most successful at earlier disease stages when CD8+ T cells are less differentiated and have greater capacity for clonotype transitions.

Mdm2/p53 levels in bone marrow mesenchymal stromal cells is essential for maintaining the hematopoietic niche in response to DNA damage.

Mesenchymal stromal cells (MSCs) are a key component of the bone marrow (BM) niche, providing essential support required for maintenance of hematopoietic stem cells. To advance our understanding of physiological functions of p53 and Mdm2 in BM-MSCs, we developed traceable conditional mouse models targeting Mdm2 and/or Trp53 in vivo . We demonstrate that Mdm2 is essential for the emergence, maintenance and hematopoietic support of BM-MSCs. Mdm2 haploinsufficiency in BM-MSCs resulted in genotoxic stress-associated thrombocytopenia, suggesting a functional role for Mdm2 in hematopoiesis. In a syngeneic mouse model of acute myeloid leukemia (AML), Trp53 deletion in BM-MSCs improved survival, and protected BM against hematopoietic toxicity from a murine Mdm2i, DS-5272. The transcriptional changes were associated with dysregulation of glycolysis, gluconeogenesis, and Hif-1α in BM-MSCs. Our results reveal a physiologic function of Mdm2 in BM-MSC, identify a previously unknown role of p53 pathway in BM-MSC-mediated support in AML and expand our understanding of the mechanism of hematopoietic toxicity of MDM2is.

Spatial Transcriptomics Depict Ligand-Receptor Cross-talk Heterogeneity at the Tumor-Stroma Interface in Long-Term Ovarian Cancer Survivors.

Advanced high-grade serous ovarian cancer (HGSC) is an aggressive disease that accounts for 70% of all ovarian cancer deaths. Nevertheless, 15% of patients diagnosed with advanced HGSC survive more than 10 years. The elucidation of predictive markers of these long-term survivors (LTS) could help identify therapeutic targets for the disease, and thus improve patient survival rates. To investigate the stromal heterogeneity of the tumor microenvironment (TME) in ovarian cancer, we used spatial transcriptomics to generate spatially resolved transcript profiles in treatment-naïve advanced HGSC from LTS and short-term survivors (STS) and determined the association between cancer-associated fibroblasts (CAF) heterogeneity and survival in patients with advanced HGSC. Spatial transcriptomics and single-cell RNA-sequencing data were integrated to distinguish tumor and stroma regions, and a computational method was developed to investigate spatially resolved ligand-receptor interactions between various tumor and CAF subtypes in the TME. A specific subtype of CAFs and its spatial location relative to a particular ovarian cancer cell subtype in the TME correlated with long-term survival in patients with advanced HGSC. Also, increased APOE-LRP5 cross-talk occurred at the stroma-tumor interface in tumor tissues from STS compared with LTS. These findings were validated using multiplex IHC. Overall, this spatial transcriptomics analysis revealed spatially resolved CAF-tumor cross-talk signaling networks in the ovarian TME that are associated with long-term survival of patients with HGSC. Further studies to confirm whether such cross-talk plays a role in modulating the malignant phenotype of HGSC and could serve as a predictive biomarker of patient survival are warranted. SIGNIFICANCE: Generation of spatially resolved gene expression patterns in tumors from patients with ovarian cancer surviving more than 10 years allows the identification of novel predictive biomarkers and therapeutic targets for better patient management. See related commentary by Kelliher and Lengyel, p. 1383.

EGFR is a master switch between immunosuppressive and immunoactive tumor microenvironment in inflammatory breast cancer.

Inflammatory breast cancer (IBC), the most aggressive breast cancer subtype, is driven by an immunosuppressive tumor microenvironment (TME). Current treatments for IBC have limited efficacy. In a clinical trial (NCT01036087), an anti-EGFR antibody combined with neoadjuvant chemotherapy produced the highest pathological complete response rate ever reported in patients with IBC having triple-negative receptor status. We determined the molecular and immunological mechanisms behind this superior clinical outcome. Using novel humanized IBC mouse models, we discovered that EGFR-targeted therapy remodels the IBC TME by increasing cytotoxic T cells and reducing immunosuppressive regulatory T cells and M2 macrophages. These changes were due to diminishing immunosuppressive chemokine expression regulated by transcription factor EGR1. We also showed that induction of an immunoactive IBC TME by an anti-EGFR antibody improved the antitumor efficacy of an anti-PD-L1 antibody. Our findings lay the foundation for clinical trials evaluating EGFR-targeted therapy combined with immune checkpoint inhibitors in patients with cancer.