Tertiary lymphoid structures and B cells determine clinically relevant T cell phenotypes in ovarian cancer.

Intratumoral tertiary lymphoid structures (TLSs) have been associated with improved outcome in various cohorts of patients with cancer, reflecting their contribution to the development of tumor-targeting immunity. Here, we demonstrate that high-grade serous ovarian carcinoma (HGSOC) contains distinct immune aggregates with varying degrees of organization and maturation. Specifically, mature TLSs (mTLS) as forming only in 16% of HGSOCs with relatively elevated tumor mutational burden (TMB) are associated with an increased intratumoral density of CD8+ effector T (TEFF) cells and TIM3+PD1+, hence poorly immune checkpoint inhibitor (ICI)-sensitive, CD8+ T cells. Conversely, CD8+ T cells from immunologically hot tumors like non-small cell lung carcinoma (NSCLC) are enriched in ICI-responsive TCF1+ PD1+ T cells. Spatial B-cell profiling identifies patterns of in situ maturation and differentiation associated with mTLSs. Moreover, B-cell depletion promotes signs of a dysfunctional CD8+ T cell compartment among tumor-infiltrating lymphocytes from freshly isolated HGSOC and NSCLC biopsies. Taken together, our data demonstrate that - at odds with NSCLC - HGSOC is associated with a low density of follicular helper T cells and thus develops a limited number of mTLS that might be insufficient to preserve a ICI-sensitive TCF1+PD1+ CD8+ T cell phenotype. These findings point to key quantitative and qualitative differences between mTLSs in ICI-responsive vs ICI-irresponsive neoplasms that may guide the development of alternative immunotherapies for patients with HGSOC.

Type I interferon signaling in malignant blasts contributes to treatment efficacy in AML patients.

While type I interferon (IFN) is best known for its key role against viral infection, accumulating preclinical and clinical data indicate that robust type I IFN production in the tumor microenvironment promotes cancer immunosurveillance and contributes to the efficacy of various antineoplastic agents, notably immunogenic cell death inducers. Here, we report that malignant blasts from patients with acute myeloid leukemia (AML) release type I IFN via a Toll-like receptor 3 (TLR3)-dependent mechanism that is not driven by treatment. While in these patients the ability of type I IFN to stimulate anticancer immune responses was abolished by immunosuppressive mechanisms elicited by malignant blasts, type I IFN turned out to exert direct cytostatic, cytotoxic and chemosensitizing activity in primary AML blasts, leukemic stem cells from AML patients and AML xenograft models. Finally, a genetic signature of type I IFN signaling was found to have independent prognostic value on relapse-free survival and overall survival in a cohort of 132 AML patients. These findings delineate a clinically relevant, therapeutically actionable and prognostically informative mechanism through which type I IFN mediates beneficial effects in patients with AML.

Polymer-ritonavir derivate nanomedicine with pH-sensitive activation possesses potent anti-tumor activity in vivo via inhibition of proteasome and STAT3 signaling.

Drug repurposing is a promising strategy for identifying new applications for approved drugs. Here, we describe a polymer biomaterial composed of the antiretroviral drug ritonavir derivative (5-methyl-4-oxohexanoic acid ritonavir ester; RD), covalently bound to HPMA copolymer carrier via a pH-sensitive hydrazone bond (P-RD). Apart from being more potent inhibitor of P-glycoprotein in comparison to ritonavir, we found RD to have considerable cytostatic activity in six mice (IC50 ~ 2.3-17.4 µM) and six human (IC50 ~ 4.3-8.7 µM) cancer cell lines, and that RD inhibits the migration and invasiveness of cancer cells in vitro. Importantly, RD inhibits STAT3 phosphorylation in CT26 cells in vitro and in vivo, and expression of the NF-κB p65 subunit, Bcl-2 and Mcl-1 in vitro. RD also dampens chymotrypsin-like and trypsin-like proteasome activity and induces ER stress as documented by induction of PERK phosphorylation and expression of ATF4 and CHOP. P-RD nanomedicine showed powerful antitumor activity in CT26 and B16F10 tumor-bearing mice, which, moreover, synergized with IL-2-based immunotherapy. P-RD proved very promising therapeutic activity also in human FaDu xenografts and negligible toxicity predetermining these nanomedicines as side-effect free nanosystem. The therapeutic potential could be highly increased using the fine-tuned combination with other drugs, i.e. doxorubicin, attached to the same polymer system. Finally, we summarize that described polymer nanomedicines fulfilled all the requirements as potential candidates for deep preclinical investigation.

M2-like macrophages dictate clinically relevant immunosuppression in metastatic ovarian cancer.

BACKGROUND: The immunological microenvironment of primary high-grade serous carcinomas (HGSCs) has a major impact on disease outcome. Conversely, little is known on the microenvironment of metastatic HGSCs and its potential influence on patient survival. Here, we explore the clinical relevance of the immunological configuration of HGSC metastases. METHODS: RNA sequencing was employed on 24 paired primary tumor microenvironment (P-TME) and metastatic tumor microenvironment (M-TME) chemotherapy-naive HGSC samples. Immunohistochemistry was used to evaluate infiltration by CD8+ T cells, CD20+ B cells, DC-LAMP+ (lysosomal-associated membrane protein 3) dendritic cells (DCs), NKp46+ (natural killer) cells and CD68+CD163+ M2-like tumor-associated macrophages (TAMs), abundance of PD-1+ (programmed cell death 1), LAG-3+ (lymphocyte-activating gene 3) cells, and PD-L1 (programmed death ligand 1) expression in 80 samples. Flow cytometry was used for functional assessments on freshly resected HGSC samples. RESULTS: 1468 genes were differentially expressed in the P-TME versus M-TME of HGSCs, the latter displaying signatures of extracellular matrix remodeling and immune infiltration. M-TME infiltration by immune effector cells had little impact on patient survival. Accordingly, M-TME-infiltrating T cells were functionally impaired, but not upon checkpoint activation. Conversely, cytokine signaling in favor of M2-like TAMs activity appeared to underlie inhibited immunity in the M-TME and poor disease outcome. CONCLUSIONS: Immunosuppressive M2-like TAM infiltrating metastatic sites limit clinically relevant immune responses against HGSCs.

Methods to assess DC-dependent priming of T cell responses by dying cells.

Dendritic cells have been widely investigated in cancer immunotherapy clinical trials for the last two decades mainly due to their robust ability to elicit an adaptive anticancer immune response of the cellular and humoral types. Immature DCs can be easily loaded with desired antigens. However, to become efficient antigen-presenting cells, DCs must first undergo a process of maturation. Protocols for the generation of DCs for use in cancer immunotherapy, including the generation of a large number of immature DCs for antigen pulsing and the selection of a well-defined immunostimulatory agent to achieve complete and reproducible maturation, which is a crucial step for further stimulation of T cell activation, must carefully consider the characteristics of DC physiology. In this report, we provided a detailed protocol for DC generation, pulsation and activation with the subsequent induction of T cell-specific immune responses.

Assessment of NK cell-mediated cytotoxicity by flow cytometry after rapid, high-yield isolation from peripheral blood.

Natural killer (NK) cells constitute the predominant innate lymphocyte subset that mediates the anti-viral and anti-tumor immune responses. NK cells use an array of innate receptors to sense their environment and to respond to infections, cellular stress and transformation. The resulting NK cell activation, including cytotoxicity and cytokine production, is a fundamental component of the early immune response. The most recent discoveries in NK cell biology have stimulated the translational research that has led to remarkable results for the treatment of human malignancies. Therefore, the rapid isolation of NK cells from the peripheral blood or tumor microenvironment and the subsequent assessment of cytolytic function are crucial to the study of their potency and NK cell-mediated immunosurveillance. Here, we provide protocols for NK cell isolation and the assessment of NK cell cytotoxicity using flow cytometry.

Calreticulin exposure correlates with robust adaptive antitumor immunity and favorable prognosis in ovarian carcinoma patients.

BACKGROUND: Adjuvanticity, which is the ability of neoplastic cells to deliver danger signals, is critical for the host immune system to mount spontaneous and therapy-driven anticancer immune responses. One of such signals, i.e., the exposure of calreticulin (CALR) on the membrane of malignant cells experiencing endoplasmic reticulum (ER) stress, is well known for its role in the activation of immune responses to dying cancer cells. However, the potential impact of CALR on the immune contexture of primary and metastatic high-grade serous carcinomas (HGSCs) and its prognostic value for patients with HGSC remains unclear. METHOD: We harnessed a retrospective cohort of primary (no = 152) and metastatic (no = 74) tumor samples from HGSC patients to investigate the CALR expression in relation with prognosis and function orientation of the tumor microenvironment. IHC data were complemented with transcriptomic and functional studies on second prospective cohort of freshly resected HGSC samples. In silico analysis of publicly available RNA expression data from 302 HGSC samples was used as a confirmatory approach. RESULTS: We demonstrate that CALR exposure on the surface of primary and metastatic HGSC cells is driven by a chemotherapy-independent ER stress response and culminates with the establishment of a local immune contexture characterized by TH1 polarization and cytotoxic activity that enables superior clinical benefits. CONCLUSIONS: Our data indicate that CALR levels in primary and metastatic HGSC samples have robust prognostic value linked to the activation of clinically-relevant innate and adaptive anticancer immune responses.

Dendritic cells pulsed with tumor cells killed by high hydrostatic pressure inhibit prostate tumor growth in TRAMP mice.

Dendritic cell (DC)-based vaccines pulsed with high hydrostatic pressure (HHP)-inactivated tumor cells have recently been shown to be a promising tool for prostate cancer chemoimmunotherapy. In this study, DC-based vaccines, both pulsed and unpulsed, were as effective as docetaxel (DTX) in reducing prostate tumors in the orthotopic transgenic adenocarcinoma of the mouse prostate (TRAMP) model. However, we did not observe any additive or synergic effects of chemoimmunotherapy on the tumor growth, while only the combination of DTX and pulsed dendritic cells resulted in significantly lower proliferation detected by Ki67 staining in histological samples. The DC-based vaccine pulsed with HHP-treated tumor cells was also combined with another type of cytostatic, cyclophosphamide, with similar results. In another clinically relevant setting, minimal residual tumor disease after surgery, administration of DC-based vaccines after the surgery of poorly immunogenic transplanted TRAMP-C2, as well as in immunogenic TC-1 tumors, reduced the growth of tumor recurrences. To identify the effector cell populations after DC vaccine application, mice were twice immunized with both pulsed and unpulsed DC vaccine, and the cytotoxicity of the spleen cells populations was tested. The effector cell subpopulations were defined as CD4+ and NK1.1+, which suggests rather unspecific therapeutic effects of the DC-based vaccines in our settings. Taken together, our data demonstrate that DC-based vaccines represent a rational tool for the treatment of human prostate cancer.

High hydrostatic pressure affects antigenic pool in tumor cells: Implication for dendritic cell-based cancer immunotherapy.

High hydrostatic pressure (HHP) can be used to generate dendritic cell (DC)-based active immunotherapy for prostate, lung and ovarian cancer. We showed here that HHP treatment of selected human cancer cell lines leads to a degradation of tumor antigens which depends on the magnitude of HHP applied and on the cancer cell line origin. Whereas prostate or ovarian cell lines displayed little protein antigen degradation with HHP treatment up to 300MPa after 2h, tumor antigens are hardly detected in lung cancer cell line after treatment with HHP 250MPa at the same time. On the other hand, quick reduction of tumor antigen-coding mRNA was observed at HHP 200MPa immediately after treatment in all cell lines tested. To optimize the DC-based active cellular therapy protocol for HHP-sensitive cell lines the immunogenicity of HHP-treated lung cancer cells at 150, 200 and 250MPa was compared. Lung cancer cells treated with HHP 150MPa display characteristics of immunogenic cell death, however cells are not efficiently phagocytosed by DC. Despite induction of the highest number of antigen-specific CD8+ T cells, 150 MPa-treated lung cancer cells survive in high numbers. This excludes their use in DC vaccine manufacturing. HHP of 200MPa treatment of lung cancer cells ensures the optimal ratio of efficient immunogenic killing and delivery of protein antigens in DC. These results represent an important pre-clinical data for generation of immunogenic killed lung cancer cells in ongoing NSCLC Phase I/II clinical trial using DC-based active cellular immunotherapy (DCVAC/LuCa).

Caspase-2 and oxidative stress underlie the immunogenic potential of high hydrostatic pressure-induced cancer cell death.

High hydrostatic pressure (HHP) promotes key characteristics of immunogenic cell death (ICD), in thus far resembling immunogenic chemotherapy and ionizing irradiation. Here, we demonstrate that cancer cells succumbing to HHP induce CD4+ and CD8+ T cell-dependent protective immunity in vivo. Moreover, we show that cell death induction by HHP relies on the overproduction of reactive oxygen species (ROS), causing rapid establishment of the integrated stress response, eIF2α phosphorylation by PERK, and sequential caspase-2, -8 and -3 activation. Non-phosphorylatable eIF2α, depletion of PERK, caspase-2 or -8 compromised calreticulin exposure by cancer cells succumbing to HHP but could not inhibit death. Interestingly, the phagocytosis of HHP-treated malignant cells by dendritic cells was suppressed by the knockdown of caspase-2 in the former. Thus, caspase-2 mediates a key function in the interaction between dying cancer cells and antigen presenting cells. Our results indicate that the ROS→PERK→eIF2α→caspase-2 signaling pathway is central for the perception of HHP-driven cell death as immunogenic.

Calreticulin exposure by malignant blasts correlates with robust anticancer immunity and improved clinical outcome in AML patients.

Cancer cell death can be perceived as immunogenic by the host only when malignant cells emit immunostimulatory signals (so-called "damage-associated molecular patterns," DAMPs), as they die in the context of failing adaptive responses to stress. Accumulating preclinical and clinical evidence indicates that the capacity of immunogenic cell death to (re-)activate an anticancer immune response is key to the success of various chemo- and radiotherapeutic regimens. Malignant blasts from patients with acute myeloid leukemia (AML) exposed multiple DAMPs, including calreticulin (CRT), heat-shock protein 70 (HSP70), and HSP90 on their plasma membrane irrespective of treatment. In these patients, high levels of surface-exposed CRT correlated with an increased proportion of natural killer cells and effector memory CD4+ and CD8+ T cells in the periphery. Moreover, CRT exposure on the plasma membrane of malignant blasts positively correlated with the frequency of circulating T cells specific for leukemia-associated antigens, indicating that ecto-CRT favors the initiation of anticancer immunity in patients with AML. Finally, although the levels of ecto-HSP70, ecto-HSP90, and ecto-CRT were all associated with improved relapse-free survival, only CRT exposure significantly correlated with superior overall survival. Thus, CRT exposure represents a novel powerful prognostic biomarker for patients with AML, reflecting the activation of a clinically relevant AML-specific immune response.

Direct culture-independent Strain typing of Burkholderia cepacia complex in sputum samples from patients with cystic fibrosis.

We examined if multilocus sequence typing (MLST), a method for genotyping and species identification of Burkholderia cepacia complex bacteria, could be applied directly to cystic fibrosis sputum. The redesigned nested-PCR MLST format was successfully used to accurately identify strains in 23 sputum samples, of which 8 were culture negative.

Oxidative stress of Burkholderia cenocepacia induces insertion sequence-mediated genomic rearrangements that interfere with macrorestriction-based genotyping.

Burkholderia cenocepacia can cause serious infections and epidemics in patients with cystic fibrosis (CF). A CF population in the Czech Republic experienced an epidemic outbreak caused by a B. cenocepacia ST-32 strain. The clonality of the isolates was evident by multilocus sequence typing; however, fingerprinting profiles obtained by pulsed-field gel electrophoresis (PFGE) showed substantial band variability. We investigated whether the PFGE pattern diversity resulted from genomic rearrangements mediated by insertion sequences (IS); in addition, we determined whether stressful growth conditions altered the transposition activity of these IS. DNA probes for IS commonly found in B. cenocepacia were designed using the B. cenocepacia J2315 genome. Southern hybridization analysis of ST-32 isolates demonstrated diversity in both the copy number and the insertion site for a homologue of ISBcen20. Movement of the ISBcen20 homologue was detected when the ST-32 isolate CZ1238 was exposed to oxidative stress (growth in the presence of H(2)O(2)). PFGE analysis of CZ1238 derivatives exposed to oxidative stress demonstrated genomic rearrangements. Interestingly, when the closely related B. cenocepacia strain J2315 was exposed to oxidative stress, no movement of ISBcen20 was detected. Since frameshift mutations are present within the transposases of all copies of this IS in J2315, our data suggest that the transposase is inactive. In summary, we have demonstrated for the first time that IS movement can be mediated by oxidative stress and can lead to genomic rearrangements in the CF pathogen B. cenocepacia. These IS movements may alter the PFGE fingerprints of isolates that are clonal by other typing methods.

High genotypic diversity of Pseudomonas aeruginosa strains isolated from patients with cystic fibrosis in the Czech Republic.

Over the last decade, epidemic and frequently multidrug-resistant Pseudomonas aeruginosa has increasingly been found among European cystic fibrosis (CF) patients. In the Czech Republic, more than half of the registered CF patients attend the Prague CF centre. At this centre, a Burkholderia cenocepacia strain was recently found to have spread among the patients. The aim of the present study was to assess whether P. aeruginosa isolates from patients at this centre were also genetically related and, if so, whether they were multidrug-resistant. We investigated a collection of 69 isolates from as many patients who represented 80% of the total number of P. aeruginosa-positive patients in 2004. The organisms were typed by AFLP and SpeI macrorestriction analyses (PFGE). Using these methods, 44 unique strains and nine groups of two to five isolates each were distinguished. Among these groups, two each had a prevalence of 7% in the patient population, while others had a prevalence of < or =3%. The diversity observed with PFGE was largely in agreement with the diversity found by AFLP analysis. All isolates were susceptible to colistin; 94-96% were susceptible to piperacillin, ceftazidime, cefepime, meropenem, amikacin or tobramycin; and 84-87% were susceptible to ciprofloxacin, gentamicin or netilmicin. In conclusion, the organisms recovered from Czech CF patients showed high genotypic diversity and good susceptibility to antipseudomonal agents. The absence of highly epidemic P. aeruginosa strains may result from infection control measures taken upon recognition of the epidemic B. cenocepacia.

Widespread clone of Burkholderia cenocepacia in cystic fibrosis patients in the Czech Republic.

The morbidity and mortality rates in patients with cystic fibrosis (CF) are significantly affected by infections with Burkholderia cepacia complex. In a Czech CF Centre, the prevalence of the infection reached up to 30 %, with the majority of patients found to be infected with Burkholderia cenocepacia (formerly genomovar III of the Burkholderia cepacia complex). Since B. cenocepacia is associated with patient-to-patient transmission and epidemic outbreaks among CF patients, this study sought to examine the epidemiological relatedness between the Czech isolates belonging to the genomovar-homogeneous group. Eighty-three clinical isolates recovered from 67 CF patients were analysed using a random amplified polymorphic DNA (RAPD) assay and macrorestriction typing (SpeI and XbaI) followed by PFGE. A single predominant banding pattern shared by multiple isolates was detected, although SpeI-generated PFGE results yielded a higher rate of inter-pattern variability in comparison to the more uniform RAPD and XbaI-generated PFGE results for this clone. Both typing systems also showed that only three out of 67 patients harboured strains distinct from the major strain type. The dominant clone was characterized by PCR positivity for the B. cepacia epidemic strain marker, PCR negativity for the cable pilin subunit gene and close genetic relatedness to the epidemic strain of RAPD 01 type previously identified in Canada.