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PUBLISHED WORK

WJH Lab Findings

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INTEGRATED T CELL CYTOMETRY METRICS FOR IMMUNE-MONITORING APPLICATIONS IN IMMUNOTHERAPY CLINICAL TRIALS

Sidiropoulos DN, Stein-O'Brien GL, Danilova L, Gross NE, Charmsaz S, Xavier S, Leatherman J, Wang H, Yarchoan M, Jaffee EM, Fertig EJ, Ho WJ. Integrated T cell cytometry metrics for immune-monitoring applications in immunotherapy clinical trials.


JCI Insight 2022

Mass cytometry, or cytometry by TOF (CyTOF), provides a robust means of determining protein-level measurements of more than 40 markers simultaneously. While the functional states of immune cells occur along continuous phenotypic transitions, cytometric studies surveying cell phenotypes often rely on static metrics, such as discrete cell-type abundances, based on canonical markers and/or restrictive gating strategies. To overcome this limitation, we applied single-cell trajectory inference and nonnegative matrix factorization methods to CyTOF data to trace the dynamics of T cell states. In the setting of cancer immunotherapy, we showed that patient-specific summaries of continuous phenotypic shifts in T cells could be inferred from peripheral blood-derived CyTOF mass cytometry data. We further illustrated that transfer learning enabled these T cell continuous metrics to be used to estimate patient-specific cell states in new sample cohorts from a reference patient data set. Our work establishes the utility of continuous metrics for CyTOF analysis as tools for translational discovery.

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PROFILING OF SYNGENEIC MOUSE HCC TUMOR MODELS AS A FRAMEWORK TO UNDERSTAND ANTI-PD-1 SENSITIVE TUMOR MICROENVIRONMENTS

Zabransky DJ, Danilova L, Leatherman JM, Lopez-Vidal TY, Sanchez J, Charmsaz S, Gross NE, Shin S, Yuan X, Hernandez A, Yang H, Xavier S, Shu D, Saeed A, Munjal K, Kamdar Z, Kagohara LT, Jaffee EM, Yarchoan M, Ho WJ

Hepatology 2022

Background and aims: The treatment of hepatocellular carcinoma (HCC) has been transformed by the use of immune checkpoint inhibitors. However, most patients with HCC do not benefit from treatment with immunotherapy. There is an urgent need to understand the mechanisms that underlie response or resistance to immunotherapy for patients with HCC. The use of syngeneic mouse models that closely recapitulate the heterogeneity of human HCC will provide opportunities to examine the complex interactions between cancer cells and nonmalignant cells in the tumor microenvironment.
Approach and results: We leverage a multifaceted approach that includes imaging mass cytometry and suspension cytometry by time of flight to profile the tumor microenvironments of the Hep53.4, Hepa 1-6, RIL-175, and TIBx (derivative of TIB-75) syngeneic mouse HCC models. The immune tumor microenvironments vary across these four models, and various immunosuppressive pathways exist at baseline in orthotopic liver tumors derived from these models. For instance, TIBx, which is resistant to anti-programmed cell death protein 1 therapy, contains a high proportion of "M2-like" tumor-associated macrophages with the potential to diminish antitumor immunity. Investigation of The Cancer Genome Atlas reveals that the baseline immunologic profiles of Hep53.4, RIL-175, and TIBx are broadly representative of human HCCs; however, Hepa 1-6 does not recapitulate the immune tumor microenvironment of the vast majority of human HCCs.
Conclusions: There is a wide diversity in the immune tumor microenvironments in preclinical models and in human HCC, highlighting the need to use multiple syngeneic HCC models to improve the understanding of how to treat HCC through immune modulation.

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NEOADJUVANT CABOZANTINIB AND NIVOLUMAB CONVERTS LOCALLY ADVANCED HCC INTO RESECTABLE DISEASE WITH ENHANCED ANTITUMOR IMMUNITY

Ho WJ, Zhu Q, Durham J, Popovic A, Xavier S, Leatherman J, Mohan A, Mo G, Zhang S, Gross N, Charmsaz S, Lin D, Quong D, Wilt B, Kamel IR, Weiss M, Philosophe B, Burkhart R, Burns WR, Shubert C, Ejaz A, He J, Deshpande A, Danilova L, Stein-O'Brien G, Sugar EA, Laheru DA, Anders RA, Fertig EJ, Jaffee EM, Yarchoan M.

Nature Cancer 2021 (Featured Cover)

A potentially curative hepatic resection is the optimal treatment for hepatocellular carcinoma (HCC), but most patients are not candidates for resection and most resected HCCs eventually recur. Until recently, neoadjuvant systemic therapy for HCC has been limited by a lack of effective systemic agents. Here, in a single arm phase 1b study, we evaluated the feasibility of neoadjuvant cabozantinib and nivolumab in patients with HCC including patients outside of traditional resection criteria (NCT03299946). Of 15 patients enrolled, 12 (80%) underwent successful margin negative resection, and 5/12 (42%) patients had major pathologic responses. In-depth biospecimen profiling demonstrated an enrichment in T effector cells, as well as tertiary lymphoid structures, CD138+ plasma cells, and a distinct spatial arrangement of B cells in responders as compared to non-responders, indicating an orchestrated B-cell contribution to antitumor immunity in HCC.

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SYSTEMIC INHIBITION OF PTPN22 AUGMENTS ANTICANCER IMMUNITY

Ho WJ, Croessmann S, Lin J, Phyo ZH, Charmsaz S, Danilova L, Mohan AA, Gross NE, Chen F, Dong J, Aggarwal D, Bai Y, Wang J, He J, Leatherman JM, Yarchoan M, Armstrong TD, Zaidi N, Fertig EJ, Denny JC, Park BH, Zhang ZY, Jaffee EM.

JCI 2021

Both epidemiologic and cellular studies in the context of autoimmune diseases have established that protein tyrosine phosphatase non-receptor type 22 (PTPN22) is a key regulator of T cell receptor (TCR) signaling. However, its mechanism of action in tumors and its translatability as a target for cancer immunotherapy have not been established. Here we show that a germline variant of PTPN22, rs2476601, portended a lower likelihood of cancer in patients. PTPN22 expression was also associated with markers of immune regulation in multiple cancer types. In mice, lack of PTPN22 augmented antitumor activity with greater infiltration and activation of macrophages, natural killer (NK) cells, and T cells. Notably, we generated a novel small molecule inhibitor of PTPN22, named L-1, that phenocopied the antitumor effects seen in genotypic PTPN22 knockout. PTPN22 inhibition promoted activation of CD8+ T cells and macrophage subpopulations toward MHC-II expressing M1-like phenotypes, both of which were necessary for successful antitumor efficacy. Increased PD1-PDL1 axis in the setting of PTPN22 inhibition could be further leveraged with PD1 inhibition to augment antitumor effects. Similarly, cancer patients with the rs2476601 variant responded significantly better to checkpoint inhibitor immunotherapy. Our findings suggest that PTPN22 is a druggable systemic target for cancer immunotherapy.

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MULTI-OMIC PROFILING OF LUNG AND LIVER TUMOR MICROENVIRONMENTS OF METASTATIC PANCREATIC CANCER REVEALS SITE-SPECIFIC IMMUNE REGULATORY PATHWAYS

Ho WJ, Erbe R, Danilova L, Phyo Z, Bigelow E, Stein-O'Brien G, Thomas DL 2nd, Charmsaz S, Gross N, Woolman S, Cruz K, Munday RM, Zaidi N, Armstrong TD, Sztein MB, Yarchoan M, Thompson ED, Jaffee EM, Fertig EJ.

Genome Biology 2021

Background: The majority of pancreatic ductal adenocarcinomas (PDAC) are diagnosed at the metastatic stage, and standard therapies have limited activity with a dismal 5-year survival rate of only 8%. The liver and lung are the most common sites of PDAC metastasis, and each have been differentially associated with prognoses and responses to systemic therapies. A deeper understanding of the molecular and cellular landscape within the tumor microenvironment (TME) metastasis at these different sites is critical to informing future therapeutic strategies against metastatic PDAC.
Results: By leveraging combined mass cytometry, immunohistochemistry, and RNA sequencing, we identify key regulatory pathways that distinguish the liver and lung TMEs in a preclinical mouse model of metastatic PDAC. We demonstrate that the lung TME generally exhibits higher levels of immune infiltration, immune activation, and pro-immune signaling pathways, whereas multiple immune-suppressive pathways are emphasized in the liver TME. We then perform further validation of these preclinical findings in paired human lung and liver metastatic samples using immunohistochemistry from PDAC rapid autopsy specimens. Finally, in silico validation with transfer learning between our mouse model and TCGA datasets further demonstrates that many of the site-associated features are detectable even in the context of different primary tumors.
Conclusions: Determining the distinctive immune-suppressive features in multiple liver and lung TME datasets provides further insight into the tissue specificity of molecular and cellular pathways, suggesting a potential mechanism underlying the discordant clinical responses that are often observed in metastatic diseases.

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A GLOBAL LIVE CELL BARCODING APPROACH FOR MULTIPLEXED MASS CYTOMETRY PROFILING OF MOUSE TUMORS

JCI Insight 2021

With the advent of cancer immunology, mass cytometry has been increasingly employed to characterize the responses to cancer therapies and the tumor microenvironment (TME). One of its most notable applications is efficient multiplexing of samples into batches by dedicating a number of metal isotope channels to barcodes, enabling robust data acquisition and analysis. Barcoding is most effective when markers are present in all cells of interest. While CD45 has been shown to be a reliable marker for barcoding all immune cells in a given sample, a strategy to reliably barcode mouse cancer cells has not been demonstrated. To this end, we identified CD29 and CD98 as markers widely expressed by commonly used mouse cancer cell lines. We conjugated anti-CD29 and anti-CD98 antibodies to cadmium or indium metals and validated their utility in 10-plex barcoding of live cells. Finally, we established a potentially novel barcoding system incorporating the combination of CD29, CD98, and CD45 to multiplex 10 tumors from s.c. MC38 and KPC tumor models, while successfully recapitulating the known contrast in the PD1-PDL1 axis between the 2 models. The ability to barcode tumor cells along with immune cells empowers the interrogation of the tumor-immune interactions in mouse TME studies.

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MULTIPANEL MASS CYTOMETRY REVEALS ANTI-PD-1 THERAPY-MEDIATED B AND T CELL COMPARTMENT REMODELING IN TUMOR-DRAINING LYMPH NODES

Ho WJ, Yarchoan M, Charmsaz S, Munday RM, Danilova L, Sztein MB, Fertig EJ, Jaffee EM.

JCI Insight 2020

Anti-programmed cell death protein 1 (anti-PD-1) therapy has become an immunotherapeutic backbone for treating many cancer types. Although many studies have aimed to characterize the immune response to anti-PD-1 therapy in the tumor and in the peripheral blood, relatively less is known about the changes in the tumor-draining lymph nodes (TDLNs). TDLNs are primary sites of tumor antigen exposure that are critical to both regulation and cross-priming of the antitumor immune response. We used multipanel mass cytometry to obtain a high-parameter proteomic (39 total unique markers) immune profile of the TDLNs in a well-studied PD-1-responsive, immunocompetent mouse model. Based on combined hierarchal gating and unsupervised clustering analyses, we found that anti-PD-1 therapy enhances remodeling of both B and T cell compartments toward memory phenotypes. Functionally, expression of checkpoint markers was increased in conjunction with production of IFN-γ, TNF-α, or IL-2 in key cell types, including B and T cell subtypes, and rarer subsets, such as Tregs and NKT cells. A deeper profiling of the immunologic changes that occur in the TDLN milieu during effective anti-PD-1 therapy may lead to the discovery of novel biomarkers for monitoring response and provide key insights toward developing combination immunotherapeutic strategies.

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