Cancer research has demonstrated that immune checkpoints have a central role in mediating immunosuppressive signalling within tumors. Immune checkpoint inhibitors, designed to block these signals, can enhance immune responses to eliminate cancer cells. Emerging evidence suggests that bacteria, similar to cancer cells, can upregulate immune checkpoints to escape immunosurveillance. Here, we explore if immune checkpoints are also upregulated in intraoperative tissue samples from periprosthetic joint infection (PJI). Paraffin-embedded periprosthetic membrane tissue from 35 patients (mean age: 69 years) undergoing revision surgery of a hip or knee joint was obtained and classified as infected (n=17) or aseptic (n=18) according to EBJIS diagnostic criteria. Tissue sections were stained with immunohistochemical protocols towards the immune checkpoints VISTA and TIM-3. A pixel classifier algorithm was trained to quantify positive immune checkpoint staining in digitally scanned tissue sections using the bioimage analysis software QuPath (Figure 1). The percentage of global VISTA staining was significantly increased in infected (mean: 1.4%, range: 0.05-5.7%) compared to aseptic revisions (mean: 0.4%, range: 0.008-1.9%), while there was no difference in TIM-3 staining. VISTA primarily stained neutrophils and lymphocytes, while TIM-3 broadly stained cells such as lymphocytes, macrophages, giant cells and fibroblasts. The percentage of VISTA and TIM-3 staining showed a significant correlation with joint type, with higher staining observed in tissue from knee compared to hip joints (r = 0.34-0.51). Increased expression of VISTA suggests that local immunosuppression is present in PJI. Therefore, it seems relevant to investigate immune checkpoints as immunotherapeutic targets for PJI. The observed heterogeneity in immune checkpoint expression among patients may reflect individual variations in immune responses during PJI. Further research on different immune checkpoints with distinct functional properties across larger patient populations is needed to understand their potential as therapeutic targets in PJI settings. Funding: Novo Nordisk Foundation and Gigtforeningen For any figures or tables, please contact the authors directly.

Cancer curing immune responses against heterogeneous solid cancers require that a coordinated immune activation is initiated in the antigen avid but immunosuppressive tumor microenvironment (TME). The plastic TME, and the poor systemic tolerability of immune activating drugs are, however, fundamental barriers to generating curative anticancer immune responses. Here, we introduce the CarboCell technology to overcome these barriers by forming an intratumoral sustained drug release depot that provides high payloads of immune stimulatory drugs selectively within the TME. The CarboCell thereby induces a hot spot for immune cell training and polarization and further drives and maintains the tumor-draining lymph nodes in an anticancer and immune activated state. Mechanistically, this transforms cancerous tissues, consequently generating systemic anticancer immunoreactivity. CarboCell can be injected through standard thin-needle technologies and has inherent imaging contrast which secure accurate intratumoral positioning. In particular, here we report the therapeutic performance for a dual-drug CarboCell providing sustained release of a Toll-like receptor 7/8 agonist and a transforming growth factor-β inhibitor in preclinical tumor models in female mice.

Liposomes carrying chemotherapeutic drugs can accumulate passively in solid tumors at high levels. However, additional targeting of the liposomes towards e.g. receptors expressed on cancer cells may improve their interaction and therapeutic properties. In this study, we designed a liposomal delivery system, which utilizes the intrinsic characteristics of HER2-positive tumors to ensure efficient delivery of oxaliplatin to the cancer cells. On the liposome surface, trastuzumab, an antibody specific to the HER2 receptor, was shown to facilitate internalization by the cancer cells. A polyethylene glycol (PEG) layer on the liposome surface provides protection from mononuclear phagocyte system uptake. To optimize the interaction between liposomes and cancer cells, a protease-sensitive cleavable peptide linker was inserted at the base of each PEG. The PEG layer is then cleaved off by intra- and extracellular matrix metalloproteinases (MMPs) upon accumulation in the tumor. Our data demonstrate that the removal of PEG significantly destabilizes the liposomes and leads to substantial oxaliplatin release. The proposed beneficial effect of combining antibody-mediated internalization with MMP sensitivity was confirmed in a series of in vivo studies using ovarian cancer xenograft models. The results demonstrated that HER2-targeted MMP-sensitive liposomes have superior anticancer activity compared to non-targeted and non-cleavable liposomes.

Cancer curing immune responses against heterogeneous solid cancers require that a coordinated immune activation is initiated in the antigen avid but immunosuppressive tumor microenvironment (TME). The plastic, immunosuppressive TME, and the poor systemic tolerability of immune activating drugs are, however, fundamental barriers to generating curative anticancer immune responses. Here, we introduce the CarboCell technology to overcome these barriers by forming a sustained drug release depot at the injection site that provides high payloads of immune stimulatory drugs selectively within the TME. The CarboCell thereby induces a hot spot for immune cell training and polarization and further drives and maintains the tumor-draining lymph nodes in an anticancer and immune activated state. Mechanistically, this transforms cancerous tissues to allow infiltration of T cells, consequently generating systemic anticancer immunoreactivity. The CarboCell technology can release multiple small molecule drugs - each with tailored release profiles - rendering it active across the broad composition of TME backgrounds. In the current study, impressive therapeutic performance is presented for a dual-drug CarboCell providing sustained release of a Toll-like receptor 7/8 agonist and a transforming growth factor-β inhibitor. CarboCell can be injected through standard clinical thin-needle technologies. Its inherent magnetic resonance imaging and ultrasound visibility, and optional radiographic contrast, make it possible to validate and plan CarboCell injections across clinical imaging modalities. These features, in combination with attractive injection intervals, secure optimal patient compliance and open new possibilities for intratumoral immunotherapy accurately across basically all anatomical locations.

Quantification of cytokines in cancerous tissue is important for understanding basic tumor biology and for deciphering anti-cancer mechanisms in drug development. Cytokine measurements on protein-level are often done by immunoassays such as enzyme-linked immunosorbent assay (ELISAs) and multiplex assays. However, immunoassays are prone to interference due to the presence of perturbing factors. The sum of these factors is known as the matrix effect, which results in a deviation of the measured cytokine concentration from the actual concentration. In this study, we demonstrated that matrix effects are present in tumor lysates from 11 different syngeneic murine tumors and that it can greatly affect cytokine measurements in ELISAs and multiplex assays. Dilution of tumor lysates and careful selection of lysis buffer components may decrease matrix effects. However, matrix effects are still present, and care should be taken when analyzing cytokine measurements of tumor lysates.

Immune-activating cytokines such as interleukin-12 (IL-12) hold strong potential for cancer immunotherapy but have been limited by high systemic toxicities. We describe here an approach to safely harness cytokine biology for adoptive cell therapy through uniform and dose-controlled tethering onto the surface of the adoptively transferred cells. Tumor-specific T cells tethered with IL-12 showed superior antitumor efficacy across multiple cell therapy models compared to conventional systemic IL-12 coadministration. Mechanistically, the IL-12–tethered T cells supported a strong safety profile by driving interferon-γ production and adoptively transferred T cell activity preferentially in the tumor. Immune profiling revealed that the tethered IL-12 reshaped the suppressive tumor immune microenvironment, including triggering a pronounced repolarization of monocytic myeloid-derived suppressor cells into activated, inflammatory effector cells that further supported antitumor activity. This tethering approach thus holds strong promise for harnessing and directing potent immunomodulatory cytokines for cell therapies while limiting systemic toxicities.

Angiogenesis is involved in regeneration of cardiac tissue following acute myocardial infarction (MI), a disease often investigated in rat models. Therefore, the ability to thoroughly evaluate the angiogenic response following experimentally induced MI in rats, and distinguish it from inflammation, is desired. This would enable evaluation of the angiogenic potential of new therapeutics and improve knowledge on MI pathophysiology. Due to the complex response to MI involving multiple cell types and the limited selection of rat-specific antibodies, careful optimization is crucial to capture this complexity. Here, we present an 8-color flow cytometry-based multicolor panel that will enable quantification of the ongoing angiogenic response as well as characterize the cells involved. A detailed description of tissue preparation, immunostaining, and gating strategy is provided. © 2021 Wiley Periodicals LLC. Basic Protocol: Cardiac tissue preparation and staining to investigate the ongoing angiogenic response in rat cardiac tissue following myocardial infarction Support Protocol: Titration of all antibodies in the presented panel.

Biomimetic high-density lipoproteins (b-HDL) have in the past two decades been applied for various drug delivery applications. As b-HDL inherently have relatively long circulation half-life and high tumor accumulation, this has inspired researchers to use b-HDL to selectively deliver drugs to tumors. PEGylation of the b-HDL has been pursued to increase the circulation half-life and therapeutic efficacy even further. The b-HDL consist of lipids stabilized by a protein/peptide scaffold, and while PEGylation of the scaffold has been shown to greatly increase the circulation half-life of the scaffold, the effect of PEGylation of the lipids is much less significant. Still, it remains to be evaluated how the biological fate, including cellular uptake, biodistribution, and circulation half-life, of the b-HDL lipids is affected by PEGylation of the b-HDL scaffold. We studied this with apolipoprotein A-I (apoA-I)-based b-HDL and mono-PEGylated b-HDL (PEG b-HDL) both in vitro and in vivo. We found that PEGylation of the b-HDL scaffold only seemed to have minimal effect on the biological fate of the lipids. Both b-HDL and PEG b-HDL overall shared similar biological fates, which includes cellular uptake through the scavenger receptor class B type 1 (SR-BI) and relatively high tumor accumulation. This highlights that b-HDL are dynamic particles, and the biological fates of the b-HDL components (lipids and scaffold) can differ. A phenomenon that may also apply for other multicomponent nanoparticles.

The tumor microenvironment (TME) composition is a critical parameter for therapeutic outcome in cancer patients. Understanding the TME composition and influence on anti-cancer therapies is a valuable tool for developing new treatments. High TME infiltration of activated tumor specific cytotoxic T-cells (cT) and central memory T-cells is crucial for improved prognosis. Conversely, interpreting infiltration of innate myeloid populations is more complex due to their plasticity and overlapping phenotypes. Myeloid derived suppressor cells (MDSC) potently inhibit cT function and proliferation in the tumor. Especially monocytic MDSCs (Mo-MDSCs) are associated with poor prognosis due to high secretion of arginase, suppressive cytokine production, and ROS-induction. Patrolling monocytes (pMos) have recently gained interest due to their regulatory function and high phagocytic capacity. Both Mo-MDSCs and pMos are progenitors to tumor-associated macrophages (TAMs), which either activate or suppress cT function. Myeloid subsets are the most prevalent infiltrating populations in both human and murine tumors and additional research is needed to provide rational selection when choosing preclinical models. In the present study, we characterized the infiltration of key myeloid subsets and activation markers in 11 commonly used syngeneic tumor models with subcutaneous tumors between 100-500 mm3. Populations of interest included; polymorphonuclear-MDSCs (PMN-MDSCs/G-MDSCs), Mo-MDSCs, TAMs, pMos, and classical dendritic cells type 1. Furthermore, we characterized key activation/regulatory markers CD80, CD86, CD163, CD206, MHCII, arginase-1, PD1, and PD-L1 to elucidate how the expression of these varies across cancer models and populations. The presented work provides a valuable tool to researcher across all categories of anti-cancer therapies by providing optimal grounds for model selection in the preclinical setting.Cell lineOriginDominant infiltrating immune population (% of viable cells)ASB-XIVPulmonary squamous cell carcinomacT (5.3%)CT26Colorectal carcinomapMo (7.1%)A20B cell lymphomaMo-MDSC (3.7%)MC38Colorectal adenocarcinomaTAM (31.4%)EG-7.OVAThymomaMo-MDSC (7.4%)SA1NFibrosacromapMo (17.6%)B16F10MelanomaMo-MDSC (3.1%)LL/2lung carcinomaMo-MDSC (17.9%)RENCARenal adenocarcinomapMo (9.2%)4T1Ductal breast carcinomaPMN-MDSC (17%)J558MyelomaVery low immune infiltrationCitation Format: Lars Ringgaard, Esben Christensen, Lotte K. Kristensen, Camilla Stavnsbjerg, Andreas Kjaer, Anders E. Hansen, Thomas L. Andresen. Characterization of the tumor microenvironment of key suppressive myeloid populations in 11 commonly used preclinical syngeneic cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1528.

Immuno-radiotherapy is a promising strategy for generation of immunologic cancer recognition, rejection and induction of anti-cancer memory. Radiotherapy (RT) causes single or double strand DNA breaks in a dose dependent manner leading to immunogenic cell death of cancer cells and induction of type 1 interferons. Furthermore, RT increases MHCI expression on cancer cells and infiltration of cytotoxic T cells. Combined, these effects make RT a potent primer of anti-cancer immune responses. The most promising approaches in anti-cancer immunotherapy is to augment the adaptive immune response through danger associated molecular patterns signaling, including toll-like receptor (TLR) agonists. Optimally, these interventions should be given when tumor-associated antigen (TAA) uptake and presentation peaks. Previous studies on TAA uptake have mostly focused on in vitro or ex vivo assays to mimic the in vivo mechanisms. In the present study, we investigate the in vivokinetics of TAA uptake and trafficking using stably transfected mCherry-transfected B16F10 melanoma. B16F10 transfected with mCherry, a pH-stable fluorescent protein, allows tracking by e.g. flow cytometry in both cancer cells and phagocytosing populations. In vivo trafficking of mCherry fluorescence was investigated in mice treated with RT alone or in combination with TLR7 agonists given locally or systemically. Following treatment, TAA-uptake, -trafficking, and activation of relevant immune populations was evaluated in tumors or tumor draining lymph nodes (tdLNs). Increased TAA uptake was observed in MHCII high populations compared to MHCII low across all phagocytosing populations in tumors. The highest uptake (MFI of mCherry) was found in patrolling monocytes (CD11b+CD11c-Ly6c-CX3CR1high) and tumor-associated macrophages (CD11b+CD11c+CD64+). Interestingly, we exclusively found increased mCherry signal in resident cDC1s in tdLNs after treatment. RT led to a diminished population of migratory CD103+ cDC1s in tumors and we did not observe any increase in mCherry+ CD103+ cDC1s in tdLNs. We did not observe any mCherry signal in spleens - indicating that no cancer-specific immune response are mounted from the spleen. The results also indicate that TAA antigen is trafficked to tdLNs primarily by monocytes or by lymphatic drainage. Citation Format: Esben Christensen, Lars Ringgaard, Anja Brus, Andreas Kjaer, Anders E. Hansen, Thomas L. Andresen. Tumor associated antigen uptake tracking following immuno-radiotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1505.