Markus A. Carlson

Targeted depletion of an MDSC subset unmasks pancreatic ductal adenocarcinoma to adaptive immunity

Background Pancreatic ductal adenocarcinoma (PDA) is characterised by a robust desmoplasia, including the notable accumulation of immunosuppressive cells that shield neoplastic cells from immune detection. Immune evasion may be further enhanced if the malignant cells fail to express high levels of antigens that are sufficiently immunogenic to engender an effector T cell response. Objective To investigate the predominant subsets of immunosuppressive cancer-conditioned myeloid cells that chronicle and shape the progression of pancreas cancer. We show that selective depletion of one subset of myeloid-derived suppressor cells (MDSC) in an autochthonous, genetically engineered mouse model (GEMM) of PDA unmasks the ability of the adaptive immune response to engage and target tumour epithelial cells. Methods A combination of in vivo and in vitro studies were performed employing a GEMM that faithfully recapitulates the cardinal features of human PDA. The predominant cancer-conditioned myeloid cell subpopulation was specifically targeted in vivo and the biological outcomes determined. Results PDA orchestrates the induction of distinct subsets of cancer-associated myeloid cells through the production of factors known to influence myelopoiesis. These immature myeloid cells inhibit the proliferation and induce apoptosis of activated T cells. Targeted depletion of granulocytic MDSC (Gr-MDSC) in autochthonous PDA increases the intratumoral accumulation of activated CD8 T cells and apoptosis of tumour epithelial cells and also remodels the tumour stroma. Conclusions Neoplastic ductal cells of the pancreas induce distinct myeloid cell subsets that promote tumour cell survival and accumulation. Targeted depletion of a single myeloid subset, the Gr-MDSC, can unmask an endogenous T cell response, disclosing an unexpected latent immunity and invoking targeting of Gr-MDSC as a potential strategy to exploit for treating this highly lethal disease.

Photostable Ratiometric Pdot Probe for in Vitro and in Vivo Imaging of Hypochlorous Acid

Developing probes for the detection of reactive oxygen species (ROS), a hallmark of many pathophysiological process, is imperative to both understanding the precise roles of ROS in many life-threatening diseases and optimizing therapeutic interventions. We herein report an all-in-one fluorescent semiconducting polymer based far-red to near-infrared (NIR) Pdot nanoprobe for the ratiometric detection of hypochlorous acid (HOCl). The fabrication takes the advantage of flexible polymer design by incorporating target-sensitive and target-inert fluorophores into a single conjugated polymer to avoid leakage or differential photobleaching problems existed in other nanoprobes. The obtained nanoprobe has improved performance in HOCl sensing, such as high brightness, ideal far-red to NIR optical window, excellent photostability, self-referenced ratiometric response, fast response, and high selectivity. The dual-emission property allows the sensitive imaging of HOCl fluctuations produced in living macrophage cells and peritonitis of living mice with high contrast. This study not only provides a powerful and promising nanoprobe to be potentially used in the investigations of in situ HOCl status of diseases in living systems but also puts forward the design strategy of a new category of ratiometric fluorescent probes facilitating precise and reliable measurement in biological systems.

Response to Chauhan et al.: Interstitial Pressure and Vascular Collapse in Pancreas Cancer—Fluids and Solids, Measurement and Meaning

Chauhan et al. suggest that vascular collapse and hypoperfusion in pancreatic ductal adenocarcinoma (PDA) are caused by solid stress (SS) (Chauhan et al., 2014) instead of the elevated interstitial fluid pressure (IFP) associated with high extravascular concentrations of hyaluronan (Provenzano et al., 2012). We appreciate their attention to our work and the opportunity to clarify underlying mechanisms. Chauhan et al. make four important claims, to which we respond.

Interstitial pressure and vascular collapse in pancreas cancer:fluids and solids, measurement and meaning

Chauhan et al. suggest that vascular collapse and hypoperfusion in pancreatic ductal adenocarcinoma (PDA) are caused by solid stress (SS) (Chauhan et al., 2014) instead of the elevated interstitial fluid pressure (IFP) associated with high extravascular concentrations of hyaluronan (Provenzano et al., 2012). We appreciate their attention to our work and the opportunity to clarify underlying mechanisms. Chauhan et al. make four important claims, to which we respond.

Abstract B05: Assessing and removing biophysical barriers to treatment

Pancreatic ductal adenocarcinoma (PDA) is a confounding cancer with a penchant for metastasis and resistance to therapy. Cell autonomous events, such as mutations in the Kras proto-oncogene, are essential for the initiation and maintenance of PDA; however non-malignant cells as well as non-cellular components have been shown to contribute to tumor growth, immunosuppression, and chemotherapeutic resistance. The stromal compartment can comprise more than 80% of tumor content and is characterized by a dynamic and deregulated extracellular matrix (ECM). In addition to a role in tissue structure, the ECM provides biophysical and biochemical cues that determine cell responses. PDA presents its own characteristic ECM signature including large deposits of the negatively charged glycosaminoglycan (GAG) hyaluronan (HA), as well as fibrillary collagens which increases matrix stiffness. We have previously demonstrated that high levels of HA in PDA contribute to extraordinary interstitial fluid pressures (IFP) and vascular collapse. We show here that IFP is comprised of both freely mobile and immobile fluid phases. Due to its highly charged nature, HA binds large amounts of water to create an immobile-fluid phase with a significant swelling pressure. This pressure is not detected by conventional methods that can measure only free fluid pressure. The swelling pressure stresses abundant collagen fibrils which contract through cellular efforts to maintain homeostasis, further contributing to IFP. Targeting HA through systemic administration of pegylated hyaluronidase (PEGPH20) liberates the immobile fluid phase and dramatically reduces IFP, increasing vessel patency and bioavailability of systemically delivered agents. We present results on the effects of targeting additional ECM components and signaling pathways to help remove biophysical barriers to chemotherapeutic access. We also compare the abilities of different methodologies to measure interstitial pressures associated with the distinct fluid phases in a variety of experimental contexts. Citation Format: Kathleen E. DelGiorno, Markus Carlson, Paolo P. Provenzano, Scott Brockenbough, Sunil R. Hingorani. Assessing and removing biophysical barriers to treatment. [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 B05.

Abstract A67: Defining signaling pathways governing metastatic potential in pancreas cancer

Pancreas cancer is an aggressively metastatic disease in which circulating tumor cells (CTC) often colonize distant organs long before diagnosis. Approximately 45% of patients present at diagnosis with metastatic disease, and this increases to 90% at the time of death. Even after surgical resection of the primary tumor, local and distant recurrence develops in approximately two thirds of cases. These sobering statistics highlight the need to define mechanisms and vulnerabilities of metastasis that can be targeted to improve patient outcomes. To better understand the process of pancreatic cancer metastasis, we study the Kras LSL-G12D/+ ; Trp53 LSL-R172H/+ ; Pdx1-Cre ( KPC ) model of autochthonous pancreas cancer. KPC mice spontaneously develop invasive and metastatic disease to numerous sites, including the liver and lungs, at rates similar to those seen in humans. Lineage tracing, performed by selectively expressing fluorescent reporters in pancreatic cells of KPC mice, has revealed a rare population of CTC that may be precursors to these metastatic foci. Building on this model, we show that alteration of TGFβ pathway components significantly affects the metastatic potential of autochthonous tumors, and that the timing and balance of signaling is critical in the metastatic process. These differences in metastatic potential are reflected in the numbers and functional properties of the respective CTC. Our distinct models with drastically different metastatic potentials provide clues to understanding the signaling mechanisms driving metastasis in pancreas cancer. Citation Format: Martin C. Whittle, Markus Carlson, Sunil R. Hingorani. Defining signaling pathways governing metastatic potential in pancreas cancer. [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 A67.

Abstract PR10: Re-engineering immunity to treat pancreas cancer

Designing T cells to effectively and safely target pancreatic ductal adenocarcinoma (PDA) has therapeutic promise, especially if combined with approaches to modify the desmoplastic and immunosuppressive tumor environment. We cloned and expressed a panel of enhanced affinity PDA-specific T cell receptors and the fate and function of such engineered tumor-specific T cells have been tracked in vivo using a genetically engineered mouse model of PDA (KPC) that we have backcrossed to C57Bl/6. Engineered T cells preferentially accumulate in PDA and express markers indicative of antigen recognition only in tumors and not peripheral sites. The transfer of engineered PDA-specific T cells significantly increased tumor cell apoptosis and remodeled the tumor matrix. However, over-time, persisting tumor-infiltrating, engineered T cells expressed markers associated with chronic antigen signaling and exhibited dysfunction as compared to donor T cells isolated from the periphery of the same host, indicating selective immunosuppression at the tumor site. Ongoing studies have identified distinct populations of immunosuppressive cells in PDA that are selectively being targeted to reveal how to sustain and enhance the activity of tumor-specific T cells. Our studies aim to elucidate the cellular and molecular signals impacting the activity of T cells specific to naturally occurring antigens overexpressed by PDA that will help to provide essential insights for the engineering of human lymphocytes and the design of clinical trials to translate our findings to patient treatment. This abstract is also presented as Poster B58. Citation Format: Ingunn M. Stromnes, Scott Brockenbrough, Thomas M. Schmitt, Jennifer D. Hotes, Markus A. Carlson, Carlos Cuevos, Philip D. Greenberg, Sunil R. Hingorani. Re-engineering immunity to treat pancreas cancer. [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 PR10.