MCMICRO: a scalable, modular image-processing pipeline for multiplexed tissue imaging.

Highly multiplexed tissue imaging makes detailed molecular analysis of single cells possible in a preserved spatial context. However, reproducible analysis of large multichannel images poses a substantial computational challenge. Here, we describe a modular and open-source computational pipeline, MCMICRO, for performing the sequential steps needed to transform whole-slide images into single-cell data. We demonstrate the use of MCMICRO on tissue and tumor images acquired using multiple imaging platforms, thereby providing a solid foundation for the continued development of tissue imaging software.

High IRF8 expression correlates with CD8 T cell infiltration and is a predictive biomarker of therapy response in ER-negative breast cancer.

BACKGROUND: Characterization of breast cancer (BC) through the determination of conventional markers such as ER, PR, HER2, and Ki67 has been useful as a predictive and therapeutic tool. Also, assessment of tumor-infiltrating lymphocytes has been proposed as an important prognostic aspect to be considered in certain BC subtypes. However, there is still a need to identify additional biomarkers that could add precision in distinguishing therapeutic response of individual patients. To this end, we focused in the expression of interferon regulatory factor 8 (IRF8) in BC cells. IRF8 is a transcription factor which plays a well-determined role in myeloid cells and that seems to have multiple antitumoral roles: it has tumor suppressor functions; it acts downstream IFN/STAT1, required for the success of some therapeutic regimes, and its expression in neoplastic cells seems to depend on a cross talk between the immune contexture and the tumor cells. The goal of the present study was to examine the relationship between IRF8 with the therapeutic response and the immune contexture in BC, since its clinical significance in this type of cancer has not been thoroughly addressed. METHODS: We identified the relationship between IRF8 expression and the clinical outcome of BC patients and validated IRF8 as predictive biomarker by using public databases and then performed in silico analysis. To correlate the expression of IRF8 with the immune infiltrate in BC samples, we performed quantitative multiplex immunohistochemistry. RESULTS: IRF8 expression can precisely predict the complete pathological response to monoclonal antibody therapy or to select combinations of chemotherapy such as FAC (fluorouracil, adriamycin, and cytoxan) in ER-negative BC subtypes. Analysis of immune cell infiltration indicates there is a strong correlation between activated and effector CD8+ T cell infiltration and tumoral IRF8 expression. CONCLUSIONS: We propose IRF8 expression as a potent biomarker not only for prognosis, but also for predicting therapy response in ER-negative BC phenotypes. Its expression in neoplastic cells also correlates with CD8+ T cell activation and infiltration. Therefore, our results justify new efforts towards understanding mechanisms regulating IRF8 expression and how they can be therapeutically manipulated.

Mucosal Immunity in the Female Murine Mammary Gland.

The mammary gland is not classically considered a mucosal organ, although it exhibits some features common to mucosal tissues. Notably, the mammary epithelium is contiguous with the external environment, is exposed to bacteria during lactation, and displays antimicrobial features. Nonetheless, immunological hallmarks predictive of mucosal function have not been demonstrated in the mammary gland, including immune tolerance to foreign Ags under homeostasis. This inquiry is important, as mucosal immunity in the mammary gland may assure infant and women's health during lactation. Further, such mucosal immune programs may protect mammary function at the expense of breast cancer promotion via decreased immune surveillance. In this study, using murine models, we evaluated mammary specific mucosal attributes focusing on two reproductive states at increased risk for foreign and self-antigen exposure: lactation and weaning-induced involution. We find a baseline mucosal program of RORγT+ CD4+ T cells that is elevated within lactating and involuting mammary glands and is extended during involution to include tolerogenic dendritic cell phenotypes, barrier-supportive antimicrobials, and immunosuppressive Foxp3+ CD4+ T cells. Further, we demonstrate suppression of Ag-dependent CD4+ T cell activation, data consistent with immune tolerance. We also find Ag-independent accumulation of memory RORγT+ Foxp3+ CD4+ T cells specifically within the involution mammary gland consistent with an active immune process. Overall, these data elucidate strong mucosal immune programs within lactating and involuting mammary glands. Our findings support the classification of the mammary gland as a temporal mucosal organ and open new avenues for exploration into breast pathologic conditions, including compromised lactation and breast cancer.

Hic-5 is required for myofibroblast differentiation by regulating mechanically dependent MRTF-A nuclear accumulation.

How mechanical cues from the extracellular environment are translated biochemically to modulate the effects of TGF-ß on myofibroblast differentiation remains a crucial area of investigation. We report here that the focal adhesion protein, Hic-5 (also known as TGFB1I1), is required for the mechanically dependent generation of stress fibers in response to TGF-ß. Successful generation of stress fibers promotes the nuclear localization of the transcriptional co-factor MRTF-A (also known as MKL1), and this correlates with the mechanically dependent induction of α smooth muscle actin (α-SMA) and Hic-5 in response to TGF-ß. As a consequence of regulating stress fiber assembly, Hic-5 is required for the nuclear accumulation of MRTF-A and the induction of α-SMA as well as cellular contractility, suggesting a crucial role for Hic-5 in myofibroblast differentiation. Indeed, the expression of Hic-5 was transient in acute wounds and persistent in pathogenic scars, and Hic-5 colocalized with α-SMA expression in vivo. Taken together, these data suggest that a mechanically dependent feed-forward loop, elaborated by the reciprocal regulation of MRTF-A localization by Hic-5 and Hic-5 expression by MRTF-A, plays a crucial role in myofibroblast differentiation in response to TGF-ß.

Mammary gland involution as an immunotherapeutic target for postpartum breast cancer.

Postpartum mammary gland involution has been identified as tumor-promotional and is proposed to contribute to the increased rates of metastasis and poor survival observed in postpartum breast cancer patients. In rodent models, the involuting mammary gland microenvironment is sufficient to induce enhanced tumor cell growth, local invasion, and metastasis. Postpartum involution shares many attributes with wound healing, including upregulation of genes involved in immune responsiveness and infiltration of tissue by immune cells. In rodent models, treatment with non-steroidal anti-inflammatory drugs (NSAIDs) ameliorates the tumor-promotional effects of involution, consistent with the immune milieu of the involuting gland contributing to tumor promotion. Currently, immunotherapy is being investigated as a means of breast cancer treatment with the purpose of identifying ways to enhance anti-tumor immune responses. Here we review evidence for postpartum mammary gland involution being a uniquely defined 'hot-spot' of pro-tumorigenic immune cell infiltration, and propose that immunotherapy should be explored for prevention and treatment of breast cancers that arise in this environment.

The group A streptococcal collagen-like protein-1, Scl1, mediates biofilm formation by targeting the extra domain A-containing variant of cellular fibronectin expressed in wounded tissue.

Wounds are known to serve as portals of entry for group A Streptococcus (GAS). Subsequent tissue colonization is mediated by interactions between GAS surface proteins and host extracellular matrix components. We recently reported that the streptococcal collagen-like protein-1, Scl1, selectively binds the cellular form of fibronectin (cFn) and also contributes to GAS biofilm formation on abiotic surfaces. One structural feature of cFn, which is predominantly expressed in response to tissue injury, is the presence of a spliced variant containing extra domain A (EDA/EIIIA). We now report that GAS biofilm formation is mediated by the Scl1 interaction with EDA-containing cFn. Recombinant Scl1 proteins that bound cFn also bound recombinant EDA within the C-C' loop region recognized by the α(9)ß(1) integrin. The extracellular 2-D matrix derived from human dermal fibroblasts supports GAS adherence and biofilm formation. Altogether, this work identifies and characterizes a novel molecular mechanism by which GAS utilizes Scl1 to specifically target an extracellular matrix component that is predominantly expressed at the site of injury in order to secure host tissue colonization.

Altered liver metabolism post-wean abolishes efficacy of vitamin D for breast cancer prevention in a mouse model.

Young women have increased risk of vitamin D deficiency, which may increase breast cancer incidence. Here, we assessed the anti-cancer efficacy of vitamin D in mouse models of young-onset breast cancer. In never-pregnant mice, vitamin D supplementation increased serum 25(OH)D and hepatic 1,25(OH)2D3, reduced tumor size, and associated with anti-tumor immunity. These anti-tumor effects were not replicated in a mouse model of postpartum breast cancer, where hepatic metabolism of vitamin D was suppressed post-wean, which resulted in deficient serum 25(OH)D and reduced hepatic 1,25(OH)2D3. Treatment with active 1,25(OH)2D3 induced hypercalcemia exclusively in post-wean mice, highlighting metabolic imbalance post-wean. RNAseq revealed suppressed CYP450 expression postpartum. In sum, we provide evidence that vitamin D anti-tumor activity is mediated through immunomodulatory mechanisms and is ineffective in the post-wean window due to altered hepatic metabolism. These findings have implications for suppressed xenobiotic metabolism in postpartum women beyond vitamin D.

Machine Learning Links T-cell Function and Spatial Localization to Neoadjuvant Immunotherapy and Clinical Outcome in Pancreatic Cancer.

Tumor molecular data sets are becoming increasingly complex, making it nearly impossible for humans alone to effectively analyze them. Here, we demonstrate the power of using machine learning (ML) to analyze a single-cell, spatial, and highly multiplexed proteomic data set from human pancreatic cancer and reveal underlying biological mechanisms that may contribute to clinical outcomes. We designed a multiplex immunohistochemistry antibody panel to compare T-cell functionality and spatial localization in resected tumors from treatment-naïve patients with localized pancreatic ductal adenocarcinoma (PDAC) with resected tumors from a second cohort of patients treated with neoadjuvant agonistic CD40 (anti-CD40) monoclonal antibody therapy. In total, nearly 2.5 million cells from 306 tissue regions collected from 29 patients across both cohorts were assayed, and over 1,000 tumor microenvironment (TME) features were quantified. We then trained ML models to accurately predict anti-CD40 treatment status and disease-free survival (DFS) following anti-CD40 therapy based on TME features. Through downstream interpretation of the ML models' predictions, we found anti-CD40 therapy reduced canonical aspects of T-cell exhaustion within the TME, as compared with treatment-naïve TMEs. Using automated clustering approaches, we found improved DFS following anti-CD40 therapy correlated with an increased presence of CD44+CD4+ Th1 cells located specifically within cellular neighborhoods characterized by increased T-cell proliferation, antigen experience, and cytotoxicity in immune aggregates. Overall, our results demonstrate the utility of ML in molecular cancer immunology applications, highlight the impact of anti-CD40 therapy on T cells within the TME, and identify potential candidate biomarkers of DFS for anti-CD40-treated patients with PDAC.

PI3Kγ inhibition circumvents inflammation and vascular leak in SARS-CoV-2 and other infections.

Virulent infectious agents such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and methicillin-resistant Staphylococcus aureus (MRSA) induce tissue damage that recruits neutrophils, monocyte, and macrophages, leading to T cell exhaustion, fibrosis, vascular leak, epithelial cell depletion, and fatal organ damage. Neutrophils, monocytes, and macrophages recruited to pathogen-infected lungs, including SARS-CoV-2-infected lungs, express phosphatidylinositol 3-kinase gamma (PI3Kγ), a signaling protein that coordinates both granulocyte and monocyte trafficking to diseased tissues and immune-suppressive, profibrotic transcription in myeloid cells. PI3Kγ deletion and inhibition with the clinical PI3Kγ inhibitor eganelisib promoted survival in models of infectious diseases, including SARS-CoV-2 and MRSA, by suppressing inflammation, vascular leak, organ damage, and cytokine storm. These results demonstrate essential roles for PI3Kγ in inflammatory lung disease and support the potential use of PI3Kγ inhibitors to suppress inflammation in severe infectious diseases.

Alignment, segmentation and neighborhood analysis in cyclic immunohistochemistry data using CASSATT.

Cyclic immunohistochemistry (cycIHC) uses sequential rounds of colorimetric immunostaining and imaging for quantitative mapping of location and number of cells of interest. Additionally, cycIHC benefits from the speed and simplicity of brightfield microscopy, making the collection of entire tissue sections and slides possible at a trivial cost compared to other high dimensional imaging modalities. However, large cycIHC datasets currently require an expert data scientist to concatenate separate open-source tools for each step of image pre-processing, registration, and segmentation, or the use of proprietary software. Here, we present a unified and user-friendly pipeline for processing, aligning, and analyzing cycIHC data - Cyclic Analysis of Single-Cell Subsets and Tissue Territories (CASSATT). CASSATT registers scanned slide images across all rounds of staining, segments individual nuclei, and measures marker expression on each detected cell. Beyond straightforward single cell data analysis outputs, CASSATT explores the spatial relationships between cell populations. By calculating the log odds of interaction frequencies between cell populations within tissues and tissue regions, this pipeline helps users identify populations of cells that interact-or do not interact-at frequencies that are greater than those occurring by chance. It also identifies specific neighborhoods of cells based on the assortment of neighboring cell types that surround each cell in the sample. The presence and location of these neighborhoods can be compared across slides or within distinct regions within a tissue. CASSATT is a fully open source workflow tool developed to process cycIHC data and will allow greater utilization of this powerful staining technique.

Machine learning links T cell function and spatial localization to neoadjuvant immunotherapy and clinical outcome in pancreatic cancer.

Tumor molecular datasets are becoming increasingly complex, making it nearly impossible for humans alone to effectively analyze them. Here, we demonstrate the power of using machine learning to analyze a single-cell, spatial, and highly multiplexed proteomic dataset from human pancreatic cancer and reveal underlying biological mechanisms that may contribute to clinical outcome. A novel multiplex immunohistochemistry antibody panel was used to audit T cell functionality and spatial localization in resected tumors from treatment-naive patients with localized pancreatic ductal adenocarcinoma (PDAC) compared to a second cohort of patients treated with neoadjuvant agonistic CD40 (αCD40) monoclonal antibody therapy. In total, nearly 2.5 million cells from 306 tissue regions collected from 29 patients across both treatment cohorts were assayed, and more than 1,000 tumor microenvironment (TME) features were quantified. We then trained machine learning models to accurately predict αCD40 treatment status and disease-free survival (DFS) following αCD40 therapy based upon TME features. Through downstream interpretation of the machine learning models' predictions, we found αCD40 therapy to reduce canonical aspects of T cell exhaustion within the TME, as compared to treatment-naive TMEs. Using automated clustering approaches, we found improved DFS following αCD40 therapy to correlate with the increased presence of CD44+ CD4+ Th1 cells located specifically within cellular spatial neighborhoods characterized by increased T cell proliferation, antigen-experience, and cytotoxicity in immune aggregates. Overall, our results demonstrate the utility of machine learning in molecular cancer immunology applications, highlight the impact of αCD40 therapy on T cells within the TME, and identify potential candidate biomarkers of DFS for αCD40-treated patients with PDAC.

Modulation of myeloid and T cells in vivo by Bruton's tyrosine kinase inhibitor ibrutinib in patients with metastatic pancreatic ductal adenocarcinoma.

BACKGROUND: In preclinical studies of pancreatic ductal adenocarcinoma (PDAC), ibrutinib improved the antitumor efficacy of the standard of care chemotherapy. This led to a phase 1b clinical trial to determine the safety, tolerability, and immunologic effects of ibrutinib treatment in patients with advanced PDAC. METHODS: Previously untreated patients with PDAC were enrolled in a phase 1b clinical trial (ClinicalTrials.gov) to determine the safety, toxicity, and maximal tolerated dose of ibrutinib when administered with the standard regimen of gemcitabine and nab-paclitaxel. To study the immune response to ibrutinib alone, the trial included an immune response arm where patients were administered with ibrutinib daily for a week followed by ibrutinib combined with gemcitabine and nab-paclitaxel. Endoscopic ultrasonography-guided primary PDAC tumor biopsies and blood were collected before and after ibrutinib monotherapy. Changes in abundance and functional state of immune cells in the blood was evaluated by mass cytometry by time of flight and statistical scaffold analysis, while that in the local tumor microenvironment (TME) were assessed by multiplex immunohistochemistry. Changes in B-cell receptor and T-cell receptor repertoire were assessed by sequencing and analysis of clonality. RESULTS: In the blood, ibrutinib monotherapy significantly increased the frequencies of activated inducible T cell costimulator+(ICOS+) CD4+ T cells and monocytes. Within the TME, ibrutinib monotherapy led to a trend in decreased B-cell abundance but increased interleukin-10+ B-cell frequency. Monotherapy also led to a trend in increased mature CD208+dendritic cell density, increased late effector (programmed cell death protein 1 (PD-1-) eomesodermin (EOMES+)) CD8+ T-cell frequency, with a concomitantly decreased dysfunctional (PD-1+ EOMES+) CD8+ T-cell frequency. When ibrutinib was combined with chemotherapy, most of these immune changes were not observed. Patients with partial clinical responses had more diverse T and B cell receptor repertoires prior to therapy initiation. CONCLUSION: Ibrutinib monotherapy skewed the immune landscape both in the circulation and TME towards activated T cells, monocytes and DCs. These effects were not observed when combining ibrutinib with standard of care chemotherapy. Future studies may focus on other therapeutic combinations that augment the immunomodulatory effects of ibrutinib in solid tumors. TRIAL REGISTRATION NUMBER: NCT02562898.

Axatilimab for Chronic Graft-Versus-Host Disease After Failure of at Least Two Prior Systemic Therapies: Results of a Phase I/II Study.

PURPOSE: Chronic graft-versus-host disease (cGVHD) remains the major cause of late morbidity after allogeneic hematopoietic cell transplantation. Colony-stimulating factor 1 receptor (CSF-1R)-dependent macrophages promote cGVHD fibrosis, and their elimination in preclinical studies ameliorated cGVHD. Axatilimab is a humanized monoclonal antibody that inhibits CSF-1R signaling and restrains macrophage development. PATIENTS AND METHODS: This phase I (phI)/phase II (phII) open-label study (ClinicalTrials.gov identifier: NCT03604692) evaluated safety, tolerability, and efficacy of axatilimab in patients age ≥ 6 years with active cGVHD after ≥ 2 prior systemic therapy lines. Primary objectives in phI were to identify the optimal biologic and recommended phII dose and in phII to evaluate the overall (complete and partial) response rate (ORR) at the start of treatment cycle 7. RESULTS: Forty enrolled patients (17 phI; 23 phII) received at least one axatilimab dose. In phI, a dose of 3 mg/kg given once every 4 weeks met the optimal biologic dose definition. Two dose-limiting toxicities occurred at the 3 mg/kg dose given once every 2 weeks. At least one treatment-related adverse event (TRAE) was observed in 30 patients with grade ≥ 3 TRAEs in eight patients, the majority known on-target effects of CSF-1R inhibition. No cytomegalovirus reactivations occurred. With the 50% ORR at cycle 7 day 1, the phII cohort met the primary efficacy end point. Furthermore, the ORR in the first six cycles, an end point supporting regulatory approvals, was 82%. Responses were seen in all affected organs regardless of prior therapy. Fifty-eight percent of patients reported significant improvement in cGVHD-related symptoms using the Lee Symptom Scale. On-target activity of axatilimab was suggested by the decrease in skin CSF-1R-expressing macrophages. CONCLUSION: Targeting profibrotic macrophages with axatilimab is a therapeutically promising novel strategy with a favorable safety profile for refractory cGVHD.

MYC Deregulation and PTEN Loss Model Tumor and Stromal Heterogeneity of Aggressive Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) patients have a poor prognosis and few treatment options. Mouse models of TNBC are important for development of new therapies, however, few mouse models represent the complexity of TNBC. Here, we develop a female TNBC murine model by mimicking two common TNBC mutations with high co-occurrence: amplification of the oncogene MYC and deletion of the tumor suppressor PTEN. This Myc;Ptenfl model develops heterogeneous triple-negative mammary tumors that display histological and molecular features commonly found in human TNBC. Our research involves deep molecular and spatial analyses on Myc;Ptenfl tumors including bulk and single-cell RNA-sequencing, and multiplex tissue-imaging. Through comparison with human TNBC, we demonstrate that this genetic mouse model develops mammary tumors with differential survival and therapeutic responses that closely resemble the inter- and intra-tumoral and microenvironmental heterogeneity of human TNBC, providing a pre-clinical tool for assessing the spectrum of patient TNBC biology and drug response.

Transforming growth factor-ß1-induced transcript 1 protein, a novel marker for smooth muscle contractile phenotype, is regulated by serum response factor/myocardin protein.

Serum response factor (SRF) plays a central role in regulating expression of smooth muscle-specific genes partly by associating with the potent tissue-specific cofactor myocardin. Previous studies have shown that transforming growth factor-ß1-induced transcript 1 (TGFB1I1, also known as Hic-5) is a TGF-ß-responsive gene and is involved in the cellular response to vascular injury, but the regulation of TGFB1I1 expression remains elusive. In this report, we demonstrated that TGFB1I1 is a novel marker for the smooth muscle contractile phenotype and is regulated by SRF/myocardin. We found that TGFB1I1 is specifically expressed in smooth muscle cells (SMCs) and in smooth muscle-rich tissues. Furthermore, TGFB1I1 expression is significantly down-regulated in a variety of models for smooth muscle phenotypic modulation. The TGFB1I1 promoter contains an evolutionarily conserved CArG element, and this element is indispensible for myocardin-induced transactivation of TGFB1I1 promoter. By oligonucleotide pulldown and chromatin immunoprecipitation assays, we found that SRF binds to this CArG element in vitro and in vivo. Ectopic expression of myocardin is sufficient to induce endogenous TGFB1I1 expression in multiple cell lines whereas knocking-down myocardin or SRF significantly attenuated TGFB1I1 expression in SMCs. Furthermore, our data demonstrated that SRF is essential for TGF-ß-mediated induction of TGFB1I1. Finally, silencing of TGFB1I1 expression significantly promotes SMC proliferation. Collectively, this study provides the first evidence that TGFB1I1 is not only an SRF/myocardin-regulated smooth muscle marker but also critical for maintaining smooth muscle contractile phenotype by inhibiting smooth muscle proliferation.

Characterization of the tumor immune microenvironment of sinonasal squamous-cell carcinoma.

BACKGROUND: Treatment and prognosis of sinonasal squamous-cell carcinoma (SNSCC) have not significantly improved despite improvements in radical therapy. Characterization of the tumor immune microenvironment (TiME) may identify patient subgroups associated with disease recurrence, and provide new biomarkers for improved patient stratification and treatment. METHODS: The TiME was quantitatively evaluated by multiplex immunohistochemistry (mIHC) in archived tissue sections from 38 patients with SNSCC, and were assessed for differences between recurrent (n = 20) and nonrecurrent (n = 18) groups. Hierarchical clustering analyses were performed to identify phenotypic TiME subgroups within the cohort and were used to compare survival outcomes. RESULTS: Our mIHC analysis revealed increased T-cell populations and decreased myeloid-cell populations in SNSCC patients without recurrent disease, as compared with patients with recurrent disease. Within T-cell subsets, there was a significantly higher percentage of granzyme B+ , T-bet+ , Eomes+ T cells, as well as higher proliferation of CD8+ T cells within the nonrecurrent group relative to the recurrent group. Furthermore, immune-cell complexity profiles of SNSCC revealed hyper- and hypo-T-cell-inflamed, myeloid-inflamed, B-cell-inflamed, and broadly hypoinflamed subtypes not previously identified by gene expression analyses. Our study revealed that presence of either hyper- or hypo-T-cell-inflamed TiME subtypes were associated with increased survival outcomes as compared with broadly hypoinflamed TiME subtypes (p = 0.035 and 0.0376, respectively). CONCLUSIONS: The TiME of SNSCC reveals distinct subtypes, which may correlate with recurrence and survival outcomes.

Quantitative Spatial Profiling of Immune Populations in Pancreatic Ductal Adenocarcinoma Reveals Tumor Microenvironment Heterogeneity and Prognostic Biomarkers.

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive disease with poor 5-year survival rates, necessitating identification of novel therapeutic targets. Elucidating the biology of the tumor immune microenvironment (TiME) can provide vital insights into mechanisms of tumor progression. In this study, we developed a quantitative image processing platform to analyze sequential multiplexed IHC data from archival PDAC tissue resection specimens. A 27-plex marker panel was employed to simultaneously phenotype cell populations and their functional states, followed by a computational workflow to interrogate the immune contextures of the TiME in search of potential biomarkers. The PDAC TiME reflected a low-immunogenic ecosystem with both high intratumoral and intertumoral heterogeneity. Spatial analysis revealed that the relative distance between IL10+ myelomonocytes, PD-1+ CD4+ T cells, and granzyme B+ CD8+ T cells correlated significantly with survival, from which a spatial proximity signature termed imRS was derived that correlated with PDAC patient survival. Furthermore, spatial enrichment of CD8+ T cells in lymphoid aggregates was also linked to improved survival. Altogether, these findings indicate that the PDAC TiME, generally considered immuno-dormant or immunosuppressive, is a spatially nuanced ecosystem orchestrated by ordered immune hierarchies. This new understanding of spatial complexity may guide novel treatment strategies for PDAC. SIGNIFICANCE: Quantitative image analysis of PDAC specimens reveals intertumoral and intratumoral heterogeneity of immune populations and identifies spatial immune architectures that are significantly associated with disease prognosis.

A Cancer Cell-Intrinsic GOT2-PPARδ Axis Suppresses Antitumor Immunity.

Despite significant recent advances in precision medicine, pancreatic ductal adenocarcinoma (PDAC) remains near uniformly lethal. Although immune-modulatory therapies hold promise to meaningfully improve outcomes for patients with PDAC, the development of such therapies requires an improved understanding of the immune evasion mechanisms that characterize the PDAC microenvironment. Here, we show that cancer cell-intrinsic glutamic-oxaloacetic transaminase 2 (GOT2) shapes the immune microenvironment to suppress antitumor immunity. Mechanistically, we find that GOT2 functions beyond its established role in the malate-aspartate shuttle and promotes the transcriptional activity of nuclear receptor peroxisome proliferator-activated receptor delta (PPARδ), facilitated by direct fatty acid binding. Although GOT2 is dispensable for cancer cell proliferation in vivo, the GOT2-PPARδ axis promotes spatial restriction of both CD4+ and CD8+ T cells from the tumor microenvironment. Our results demonstrate a noncanonical function for an established mitochondrial enzyme in transcriptional regulation of immune evasion, which may be exploitable to promote a productive antitumor immune response. SIGNIFICANCE: Prior studies demonstrate the important moonlighting functions of metabolic enzymes in cancer. We find that the mitochondrial transaminase GOT2 binds directly to fatty acid ligands that regulate the nuclear receptor PPARδ, and this functional interaction critically regulates the immune microenvironment of pancreatic cancer to promote tumor progression. See related commentary by Nwosu and di Magliano, p. 2237.. This article is highlighted in the In This Issue feature, p. 2221.

Safety and Efficacy of Pembrolizumab in Combination with Acalabrutinib in Advanced Head and Neck Squamous Cell Carcinoma: Phase 2 Proof-of-Concept Study.

PURPOSE: Programmed cell death-1 (PD-1) receptor inhibitors have shown efficacy in head and neck squamous cell carcinoma (HNSCC), but treatment failure or secondary resistance occurs in most patients. In preclinical murine carcinoma models, inhibition of Bruton's tyrosine kinase (BTK) induces myeloid cell reprogramming that subsequently bolsters CD8+ T cell responses, resulting in enhanced antitumor activity. This phase 2, multicenter, open-label, randomized study evaluated pembrolizumab (anti-PD-1 monoclonal antibody) plus acalabrutinib (BTK inhibitor) in recurrent or metastatic HNSCC. PATIENTS AND METHODS: Patients received pembrolizumab 200 mg intravenously every 3 weeks, alone or in combination with acalabrutinib 100 mg orally twice daily. Safety and overall response rate (ORR) were co-primary objectives. The secondary objectives were progression-free survival (PFS) and overall survival. RESULTS: Seventy-six patients were evaluated (pembrolizumab, n = 39; pembrolizumab + acalabrutinib, n = 37). Higher frequencies of grade 3-4 treatment-emergent adverse events (AE; 65% vs. 39%) and serious AEs (68% vs. 31%) were observed with combination therapy versus monotherapy. ORR was 18% with monotherapy versus 14% with combination therapy. Median PFS was 2.7 [95% confidence interval (CI), 1.4-6.8] months in the combination arm and 1.7 (95% CI, 1.4-4.0) months in the monotherapy arm. The study was terminated due to lack of clinical benefit with combination treatment. To assess how tumor immune contexture was affected by therapy in patients with pre- and post-treatment biopsies, spatial proteomic analyses were conducted that revealed a trend toward increased CD45+ leukocyte infiltration of tumors from baseline at day 43 with pembrolizumab (monotherapy, n = 5; combination, n = 2), which appeared to be higher in combination-treated patients; however, definitive conclusions could not be drawn due to limited sample size. CONCLUSIONS: Despite lack of clinical efficacy, immune subset analyses suggest that there are additive effects of this combination; however, the associated toxicity limits the feasibility of combination treatment with pembrolizumab and acalabrutinib in patients with recurrent or metastatic HNSCC.