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Background: Neoadjuvant immune checkpoint inhibition (ICI) in combination with chemotherapy is approved for patients with high-risk, early-stage triple-negative breast cancer (TNBC) based on improved outcomes in the KEYNOTE-522 trial. However, some patients have primary resistant disease and do not achieve a pathological complete response (pCR), while others experience significant toxicity. Tumor-associated macrophages (TAMs) are a potential resistance mechanism for ICIs and are dependent on colony-stimulating factor 1 receptor (CSF1R). Therefore, we examined the addition of cabiralizumab, a CSF1R inhibitor, to neoadjuvant paclitaxel, carboplatin, and nivolumab to assess the safety, tolerability, and changes in the tumor microenvironment (TME) in patients with early-stage TNBC. Methods: This is a phase Ib/II, single-institution, randomized controlled clinical trial (NCT04331067) in patients with newly diagnosed Stage II-III TNBC. The primary endpoints include: (1) to determine the safety of a 12-week neoadjuvant regimen of paclitaxel (80 mg/m2 IV q week) + carboplatin (AUC5 IV q3 weeks) + nivolumab (240 mg IV q2 weeks) with or without cabiralizumab (4 mg/kg IV q2 weeks) and (2) to evaluate the effect of cabiralizumab on TAMs and changes in tumor infiltrating lymphocytes (TILs) in the TME between baseline and an on-treatment biopsy after 4 weeks of therapy. Adjuvant treatment is per investigator’s choice. Secondary objectives include evaluation of pCR rate and recurrence-free survival. Paired tissue and bone marrow biopsies are collected for evaluation of the TME and disseminated tumor cells, respectively. The study was designed to enroll 50 patients, including a 12-patient safety lead-in cohort. Here, we report the planned interim analysis of the safety lead-in cohort. Results: Between December 2020 and May 2022, we enrolled 12 patients to the safety lead-in, including 6 patients in each arm. 5 of 12 patients (41.7%) enrolled are underrepresented minorities, including 4 Black patients and 1 Hispanic patient. 2 of 6 patients in the nivolumab arm experienced grade 3 severe toxicity, including 1 patient who developed sepsis and 1 who developed peripheral neuropathy. 3 of 6 patients in the nivolumab + cabiralizumab arm developed grade 3 severe toxicity including 2 patients who experienced myositis and 1 patient who developed periorbital edema. Of the first 10 patients enrolled, 5 had a pCR (2 pCR in cabiralizumab arm, 3 pCR in non-cabiralizumab arm) and 3 had non-pCR (1 RCB-1 and 1 RCB-3 in cabiralizumab arm, 1 RCB-1 in non-cabiralizumab arm). 2 patients came off study prior to surgery (1 due to toxicity and 1 due to missing study visits). Data from the final 2 patients still on treatment will be available at the time of presentation. Discussion: Full safety, pathologic, and clinical response data in the safety lead-in cohort for patients with early-stage TNBC receiving neoadjuvant chemotherapy + nivolumab with or without cabiralizumab, will be presented. Citation Format: Andrew A. Davis, Leonel Hernandez-Aya, Jingqin Luo, Mateusz Opyrchal, Foluso O. Ademuyiwa, Nusayba A. Bagegni, Katherine K. Clifton, Jill Anderson, Trish Hammerschmidt, Leslie Nehring, David DeNardo, Mark Watson, Rebecca Aft, Cynthia Ma, Katherine Weilbaecher. Phase Ib/II study to evaluate safety and tolerability of cabiralizumab in combination with nivolumab and neoadjuvant chemotherapy in patients with localized triple-negative breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P3-06-07.

Numerous cell states are known to comprise the pancreatic ductal adenocarcinoma (PDAC) tumor microenvironment (TME). However, the developmental stemness and co-occurrence of these cell states remain poorly defined. Here, we performed single-cell RNA sequencing (scRNA-seq) on a cohort of treatment-naive PDAC time-of-diagnosis endoscopic ultrasound-guided fine needle biopsy (EUS-FNB) samples (n=25) and surgical resections (n=6), which were then combined with publicly available samples to increase statistical power (n=80). Following annotation into 25 distinct cell states, cells were scored for developmental stemness and a customized version of the Ecotyper tool was used to identify communities of co-occurring cell states in bulk RNA-seq samples (n=268). We discovered a tumor microenvironmental community comprised of aggressive basal-like malignant cells, tumor-promoting SPP1+ macrophages, and myofibroblastic cancer-associated fibroblasts associated with especially poor prognosis. We also found a developmental stemness continuum with implications for survival that is present in both malignant cells and cancer-associated fibroblasts (CAFs). We further demonstrated that high-dimensional analyses predictive of survival are feasible using standard-of-care, time-of-diagnosis EUS-FNB specimens. In summary, we identified novel tumor microenvironmental and developmental stemness characteristics from a high-dimensional gene expression analysis of PDAC using human tissue specimens including time-of-diagnosis EUS-FNB samples. These reveal new connections between tumor microenvironmental composition, CAF and malignant cell stemness, and patient survival that could lead to better upfront risk stratification and more personalized upfront clinical decision-making.

Retinoids have been found to be promising chemopreventive agents that play an important role in regulating cell growth, differentiation, and apoptosis. The action of retinoids is mediated by retinoid receptors (retinoic acid receptors and retinoid X receptors), which are nuclear transcription factors that, when bound to retinoids, regulate gene expression. LGD1069 is a highly selective RXR agonist that has reduced toxicity compared with retinoids. Our previous studies have shown that RXR-selective ligands (or “rexinoids”), including LGD1069, can inhibit the growth of normal and malignant breast cells and can suppress the development of breast cancer in transgenic mice. For the current study, we attempted to identify biomarkers of the chemopreventive effect of the RXR-selective retinoid LGD1069. In these experiments, we used Affymetrix microarrays to identify target genes that were modulated by LGD1069 in normal human breast cells. Affymetrix and dChip analysis identified more than 100 genes that were up-regulated or down-regulated by LGD1069 treatment. We then tested 16 of these genes in validation experiments using quantitative reverse transcription-PCR and Western blotting of independently prepared samples, and found that 15 of 16 genes were modulated in a similar manner in these validation experiments as in the microarray experiments. Genes found to be regulated include known retinoid-regulated genes, growth regulatory genes, transcription factors, and differentiation markers. We then showed that the expression of several of these rexinoid-regulated biomarkers is modulated in vivo in mammary glands from mice treated with LGD1069. These critical growth-regulating proteins will be promising targets of future agents for the prevention and treatment of breast cancer. (Cancer Res 2006; 66(24): 12009-18)

The effects of nab-PXL on upregulation of cytokines in MDA-MB-231 cells expressing dominant negative (DN)-TLR4 or DN-MyD88 and control cell lines (S1); The comparison of nab-PXL and docetaxel effects on the TLR4-positive human and mouse BC cells (S2); FACS analysis of spleen and bone marrow cells (S3); Effect of a blocking mouse CD11b antibody on PXL-induced tumor recruitment of myeloid cells and blood vascular densities (S4).

Supplementary Figures 1-9 from Receptor Tyrosine Kinase Signaling Favors a Protumorigenic State in Breast Cancer Cells by Inhibiting the Adaptive Immune Response

Legend for Supplementary Figures S1-S4.

Supplementary Diagrams 1-2 from Identification of Biomarkers Modulated by the Rexinoid LGD1069 (Bexarotene) in Human Breast Cells Using Oligonucleotide Arrays

Supplementary Figures 3-5 from Identification of Biomarkers Modulated by the Rexinoid LGD1069 (Bexarotene) in Human Breast Cells Using Oligonucleotide Arrays

Supplementary Tables 1-6 from Receptor Tyrosine Kinase Signaling Favors a Protumorigenic State in Breast Cancer Cells by Inhibiting the Adaptive Immune Response

Supplementary Tables 1-4, Figures 1-2 from Identification of Biomarkers Modulated by the Rexinoid LGD1069 (Bexarotene) in Human Breast Cells Using Oligonucleotide Arrays

RT-qPCR Primers (S1); Effect of TLR4 on incidence and metastatic burden in nab-PXL treated and untreated mice (S2).

Supplementary Methods and Figure Legends 1-13 from Receptor Tyrosine Kinase Signaling Favors a Protumorigenic State in Breast Cancer Cells by Inhibiting the Adaptive Immune Response

T cells engineered to express chimeric antigen receptors (CARs) targeting CD19 have demonstrated impressive activity against relapsed or refractory B cell cancers yet fail to induce durable remissions for nearly half of patients treated. Enhancing the efficacy of this therapy requires detailed understanding of the molecular circuitry that restrains CAR-driven anti-tumor T cell function. We developed and validated an in vitro model that drives T cell dysfunction through chronic CAR activation and interrogated how CAR costimulatory domains, central components of CAR structure and function, contribute to T cell failure. We found that chronic activation of CD28-based CARs results in activation of classical T cell exhaustion programs and development of dysfunctional cells that bear the hallmarks of exhaustion. In contrast, 41BB-based CARs activate a divergent molecular program and direct differentiation of T cells into a novel cell state. Interrogation of CAR T cells from a patient with progressive lymphoma confirmed activation of this novel program in a failing clinical product. Further, we demonstrate that 41BB-dependent activation of the transcription factor FOXO3 is directly responsible for impairing CAR T cell function. These findings identify that costimulatory domains are critical regulators of CAR-driven T cell failure and that targeted interventions are required to overcome costimulation-dependent dysfunctional programs.

Severe and prolonged lymphopenia frequently occurs in patients with glioblastoma after standard chemoradiotherapy and has been associated with worse survival, but its underlying biological mechanism is not well understood. To address this, we performed a correlative study in which we collected and analyzed peripheral blood of patients with glioblastoma ( n = 20) receiving chemoradiotherapy using genomic and immune monitoring technologies. RNA sequencing analysis of the peripheral blood mononuclear cells (PBMC) showed an elevated concentration of myeloid-derived suppressor cell (MDSC) regulatory genes in patients with lymphopenia when compared with patients without lymphopenia after chemoradiotherapy. Additional analysis including flow cytometry and single-cell RNA sequencing further confirmed increased numbers of circulating MDSC in patients with lymphopenia when compared with patients without lymphopenia after chemoradiotherapy. Preclinical murine models were also established and demonstrated a causal relationship between radiation-induced MDSC and systemic lymphopenia using transfusion and depletion experiments. Pharmacological inhibition of MDSC using an arginase-1 inhibitor (CB1158) or phosphodiesterase-5 inhibitor (tadalafil) during radiation therapy (RT) successfully abrogated radiation-induced lymphopenia and improved survival in the preclinical models. CB1158 and tadalafil are promising drugs in reducing radiation-induced lymphopenia in patients with glioblastoma. These results demonstrate the promise of using these classes of drugs to reduce treatment-related lymphopenia and immunosuppression.

SummaryB cells are critical for adaptive immunity and are governed by the recognition of an antigen by the B cell receptor (BCR), a process that drives a coordinated series of signaling events and modulation of various transcriptional programs. Single-cell RNA sequencing with paired BCR profiling could offer insights into numerous physiological and pathological processes. However, unlike the plethora of single-cell RNA analysis pipelines, computational tools that utilize single-cell BCR sequences for further analyses are not yet well developed. Here we report Ibex, which vectorizes the amino acid sequence of the complementarity-determining region 3 (cdr3) of the immunoglobulin heavy and light chains, allowing for unbiased dimensional reduction of B cells using their BCR repertoire. Ibex is implemented as an R package with integration into both the Seurat and Single-Cell Experiment framework, enabling the incorporation of this new analytic tool into many single-cell sequencing analytic workflows and multimodal experiments.Availability and ImplementationIbex is available as an R package athttps://github.com/ncborcherding/Ibex. Reproducible code and data for the figure appearing in the manuscript are available athttps://github.com/ncborcherding/Ibex.manuscript. A companion TCR-based approach is available athttps://github.com/ncborcherding/Trex.

Chronic activation of inflammatory pathways and suppressed interferon signaling are hallmarks of myeloid cells in immunosuppressive tumors that drive poor responsiveness to conventional and immune therapies. Previous studies have identified agonistic activation of the CD11b integrin as a potential strategy to enhance anti-tumor immunity. However, the mechanisms by which CD11b-agonism reprogram tumor immunity are poorly understood, and this may impair patient selection and identification of effective treatment combinations. Here we will use a combination of in vitro systems, animal models, and samples from first in human clinical trials of the CD11b-agonist to identify the mechanism of action of this approach and identify combinations for further testing. CD11b agonism alters tumor-associated macrophage (TAM) phenotypes by simultaneously repressing NFκB/IL1-signaling and activating interferon (IFN) gene expression. Repression of NFκB/IL-1 signaling was due to rapid degradation of p65 protein by the proteosome, contributing to suppressing myeloid cells infiltration. In contrast, CD11b agonism triggers mitochondrial dysfunction to stimulate STING-induced, STAT1-mediated interferon signaling, contributing to macrophages-augmented anti-tumor T cell immunity. Tissues from phase I clinical trials shows that CD11b agonist activates STING and STAT1 signaling in TAMs in human tumors, which is in consistent with preclinical data. Furthermore, combining CD11b agonism with cytotoxic therapies, STING agonist or TLRs agonists leads to robust anti-tumor activity in PDAC models. These studies pave the way for mechanism-based therapeutic combination strategies in cancer patients. Citation Format: Xiuting Liu, Graham Hogg, Chong Zuo, John Baer, Varintra Lander, Liang Kang, Nicholas Borcherding, Brett Knolhoff, Robin Osterhout, Anna Galkin, Jean Bruey, Laura Carter, Cedric Mpoy, Julie Schwarz, Haeseong Park, Vineet Gupta, David DeNardo. Context-dependent induction of interferon signaling by CD11b agonists supports anti-tumor immunity in mouse models and human cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5964.

The philosophy, design and technical aspects of Shark Experience, an exhibit that was created to educate the public about sharks and the importance of their conservation, are outlined in this paper. Details are given of the physical aspects of the exhibit, the specimens selected and a description of the methods of disseminating information about sharks and their role in the natural world.

The immune system is a promising therapeutic target that can be modulated to improve pancreatic ductal adenocarcinoma (PDAC) patient outcomes. Unfortunately, attempts to use immunotherapeutics as single agents have achieved very limited clinical benefits in PDAC. One major obstacle is the presence of an immunosuppressive tumor microenvironment. Critical drivers of this immunosuppressive microenvironment include tumor-associated macrophages, monocytic myeloid-derived suppressor cells, and immature granulocytes/granulocytic. In addition to instigating immune suppression, these tumor-infiltrating leukocytes can impart resistance to cytotoxic therapies and promote metastatic dissemination. As a consequence, high numbers of these myeloid cells are often associated with early metastatic relapse and poor survival in pancreatic cancer patients. Therefore, approaches that reprogram myeloid responses to potentiate antitumor immunity and/or bolster immunotherapy could hold significant therapeutic potential. In this report, we demonstrate that inhibition of colony stimulating factor 1 receptor (CSF1R) signaling functionally reprograms macrophage responses to enhance antigen presentation, thereby stimulating anti-tumor T cell responses. However, this immune reprograming also leads to the upregulation of T cell checkpoint molecules, including PDL1 and CTLA4, which significantly limits the potential of the CSF1R blockade to induce anti-tumor responses. While both programmed cell death protein 1 and CTLA4 antagonists show limited efficacy as single agents to restrain PDAC growth in a mouse model, the combination of these agents with CSF1R blockade achieves disease regression even in well-established tumors. These data suggest that reprograming myeloid responses through CSF1R blockade can improve the efficacy of checkpoint-based immunotherapeutics in pancreatic cancer. Citation Format: Yu Zhu, Knolhoff Brett, Meyer Melissa, West Brian, Linehan David, David DeNardo. CSF1R blockade reprograms tumor-infiltrating macrophages and improves response to T-cell checkpoint immunotherapy in pancreatic cancer models. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr B61.