Although current immune-checkpoint therapy (ICT) mainly targets lymphoid cells, it is associated with a broader remodeling of the tumor micro-environment. Here, using complementary forms of ...high-dimensional profiling, we define differences across all hematopoietic cells from syngeneic mouse tumors during unrestrained tumor growth or effective ICT. Unbiased assessment of gene expression of tumor-infiltrating cells by single-cell RNA sequencing (scRNAseq) and longitudinal assessment of cellular protein expression by mass cytometry (CyTOF) revealed significant remodeling of both the lymphoid and myeloid intratumoral compartments. Surprisingly, we observed multiple subpopulations of monocytes/macrophages, distinguishable by the markers CD206, CX3CR1, CD1d, and iNOS, that change over time during ICT in a manner partially dependent on IFNγ. Our data support the hypothesis that this macrophage polarization/activation results from effects on circulatory monocytes and early macrophages entering tumors, rather than on pre-polarized mature intratumoral macrophages.
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•High-dimensional analyses of successful ICT in tumor-bearing mice•ICT induces changes in intratumoral myeloid and lymphoid cells•Tumor-associated monocytes/macrophages display complex cytokine-driven phenotypes•Different cytokines act on tumor-infiltrating monocytes to drive macrophage polarization
Comprehensive changes in the tumor microenvironment during successful immune-checkpoint therapy are profiled, implicating a key role for polarization of infiltrating macrophages in the anti-tumor immune milieu.
Advances from glioma stemlike cell (GSC) research, though increasing our knowledge of glioblastoma (GBM) biology, do not influence clinical decisions yet. We explored the translational power of ...GSC-enriched cultures from patient-derived tumorspheres (TS) in predicting treatment response.
The relationship between TS growth and clinical outcome was investigated in 52 GBMs treated with surgical resection followed by radiotherapy and temozolomide (TMZ). The effect on TS of radiation (6 to 60 Gy) and of TMZ (3.9 μM to 1 mM) was related with patients' survival.
Generation of TS was an independent factor for poor overall survival (OS) and poor progression-free survival (PFS) (P < .0001 and P = .0010, respectively). Growth rate and clonogenicity of TS predicted poor OS. In general, TS were highly resistant to both radiation and TMZ. Resistance to TMZ was stronger in TS with high clonogenicity and fast growth (P < .02). Shorter PFS was associated with radiation LD50 (lethal dose required to kill 50% of TS cells) >12 Gy of matched TS (P = .0484). A direct relationship was found between sensitivity of TS to TMZ and patients' survival (P = .0167 and P = .0436 for OS and PFS, respectively). Importantly, values for TMZ half-maximal inhibitory concentration <50 μM, which are in the range of plasma levels achieved in vivo, identified cases with longer OS and PFS (P = .0020 and P = .0016, respectively).
Analysis of TS holds translational relevance by predicting the response of parent tumors to radiation and, particularly, to TMZ. Dissecting the clonogenic population from proliferating progeny in TS can guide therapeutic strategies to a more effective drug selection and treatment duration.
Chemoresistance of glioblastoma multiforme (GBM) has been attributed to the presence within the tumor of cancer stem cells (GSCs). The standard therapy for GBM consists of surgery followed by ...radiotherapy and the chemotherapeutic agent temozolomide (TMZ). However, TMZ efficacy is limited by O6-methylguanine-DNA-methyltransferase (MGMT) and Mismatch Repair (MMR) functions. Strategies to counteract TMZ resistance include its combination with poly(ADP-ribose) polymerase inhibitors (PARPi), which hamper the repair of N-methylpurines. PARPi are also investigated as monotherapy for tumors with deficiency of homologous recombination (HR). We have investigated whether PARPi may restore GSC sensitivity to TMZ or may be effective as monotherapy.
Ten human GSC lines were assayed for MMR proteins, MGMT and PARP-1 expression/activity, MGMT promoter methylation and sensitivity to TMZ or PARPi, alone and in combination. Since PTEN defects are frequently detected in GBM and may cause HR dysfunction, PTEN expression was also analyzed. The statistical analysis of the differences in drug sensitivity among the cell lines was performed using the ANOVA and Bonferroni's post-test or the non-parametric Kruskal-Wallis analysis and Dunn's post-test for multiple comparisons. Synergism between TMZ and PARPi was analyzed by the median-effect method of Chou and Talalay. Correlation analyses were done using the Spearman's rank test.
All GSCs were MMR-proficient and resistance to TMZ was mainly associated with high MGMT activity or low proliferation rate. MGMT promoter hypermethylation of GSCs correlated both with low MGMT activity/expression (Spearman's test, P = 0.004 and P = 0.01) and with longer overall survival of GBM patients (P = 0.02). Sensitivity of each GSC line to PARPi as single agent did not correlate with PARP-1 or PTEN expression. Notably, PARPi and TMZ combination exerted synergistic antitumor effects in eight out of ten GSC lines and the TMZ dose reduction achieved significantly correlated with the sensitivity of each cell line to PARPi as single agent (P = 0.01).
The combination of TMZ with PARPi may represent a valuable strategy to reverse GSC chemoresistance.
Estrogen receptor alpha-positive (ERα+) luminal tumors are the most frequent subtype of breast cancer. Stat1−/− mice develop mammary tumors that closely recapitulate the biological characteristics of ...this cancer subtype. To identify transforming events that contribute to tumorigenesis, we performed whole genome sequencing of Stat1−/− primary mammary tumors and matched normal tissues. This investigation identified somatic truncating mutations affecting the prolactin receptor (PRLR) in all tumor and no normal samples. Targeted sequencing confirmed the presence of these mutations in precancerous lesions, indicating that this is an early event in tumorigenesis. Functional evaluation of these heterozygous mutations in Stat1−/− mouse embryonic fibroblasts showed that co-expression of truncated and wild-type PRLR led to aberrant STAT3 and STAT5 activation downstream of the receptor, cellular transformation in vitro, and tumor formation in vivo. In conclusion, truncating mutations of PRLR promote tumor growth in a model of human ERα+ breast cancer and warrant further investigation.
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•Mice lacking Stat1 develop mammary tumors that model human ERα+ breast cancer•DNA sequencing finds a Prlr truncating mutation hotspot in 100% of Stat1−/− tumors•At least 77.8% of DCIS examined also contain PRLR truncating mutations•Co-expression of truncated and full-length PRLR promote tumor growth in MEFs
Griffith et al. report on whole genome sequencing of mammary tumors that spontaneously develop in mice lacking Stat1. They find a 100% recurrent hotspot of mutations resulting in truncation of Prlr. Co-expression of truncated and full-length Prlr in mouse embryonic fibroblasts is shown to promote tumor growth.
The ability of the immune system to eliminate and shape the immunogenicity of tumours defines the process of cancer immunoediting
. Immunotherapies such as those that target immune checkpoint ...molecules can be used to augment immune-mediated elimination of tumours and have resulted in durable responses in patients with cancer that did not respond to previous treatments. However, only a subset of patients benefit from immunotherapy and more knowledge about what is required for successful treatment is needed
. Although the role of tumour neoantigen-specific CD8
T cells in tumour rejection is well established
, the roles of other subsets of T cells have received less attention. Here we show that spontaneous and immunotherapy-induced anti-tumour responses require the activity of both tumour-antigen-specific CD8
and CD4
T cells, even in tumours that do not express major histocompatibility complex (MHC) class II molecules. In addition, the expression of MHC class II-restricted antigens by tumour cells is required at the site of successful rejection, indicating that activation of CD4
T cells must also occur in the tumour microenvironment. These findings suggest that MHC class II-restricted neoantigens have a key function in the anti-tumour response that is nonoverlapping with that of MHC class I-restricted neoantigens and therefore needs to be considered when identifying patients who will most benefit from immunotherapy.
Significance
Immune checkpoint therapy (ICT) has led to durable responses in a subset of cancer patients. Generally, patients who respond to ICT bear tumors with high mutational burden. Radiation is ...used for treatment of many types of cancers and has been shown to induce new mutations in treated tumor cells and to synergistically facilitate ICT. However, these latter actions have largely been explained by radiation-induced tumor cell death and/or effects on the host. Herein, we show that noncurative irradiation induces mutations in tumor cells lacking neoantigens and that these de novo-generated neoantigens function as targets for CD8
+
T cells, resulting in increased immunogenicity of nonimmunogenic tumor cells. This study thus identifies an additional mechanism that explains synergy between immunotherapy and radiation.
Immunotherapies are a promising advance in cancer treatment. However, because only a subset of cancer patients benefits from these treatments it is important to find mechanisms that will broaden the responding patient population. Generally, tumors with high mutational burdens have the potential to express greater numbers of mutant neoantigens. As neoantigens can be targets of protective adaptive immunity, highly mutated tumors are more responsive to immunotherapy. Given that external beam radiation 1) is a standard-of-care cancer therapy, 2) induces expression of mutant proteins and potentially mutant neoantigens in treated cells, and 3) has been shown to synergize clinically with immune checkpoint therapy (ICT), we hypothesized that at least one mechanism of this synergy was the generation of de novo mutant neoantigen targets in irradiated cells. Herein, we use Kras
G12D
x p53
−/−
sarcoma cell lines (KP sarcomas) that we and others have shown to be nearly devoid of mutations, are poorly antigenic, are not controlled by ICT, and do not induce a protective antitumor memory response. However, following one in vitro dose of 4- or 9-Gy irradiation, KP sarcoma cells acquire mutational neoantigens and become sensitive to ICT in vivo in a T cell-dependent manner. We further demonstrate that some of the radiation-induced mutations generate cytotoxic CD8
+
T cell responses, are protective in a vaccine model, and are sufficient to make the parental KP sarcoma line susceptible to ICT. These results provide a proof of concept that induction of new antigenic targets in irradiated tumor cells represents an additional mechanism explaining the clinical findings of the synergy between radiation and immunotherapy.
With the beginning of the new millennium
intravital
multiphoton imaging of the immune system has become a necessary tool for understanding cellular behavior in steady state and models of disease,
in ...vivo
. The first studies visualized dynamics of antigen presenting cells and T‐cells. Followed by development of fluorescent reporter mice that allowed tracking of neutrophils, monocytes and macrophages, however the spatiotemporal resolution achieved wasn't sufficient enough to study physiological parameters like blood or lymph flow.
We have improved our two‐photon imaging approach by decreasing photo toxicity and generated a combination of fluorophores which are low in cytotoxicity, able to label morphological structures and cell populations of interest. This combination of dyes is based on high excitation efficiency capable to be excited with low laser power and bright emission properties. Together with our customized dual femtosecond pulsed laser system (InSight DS+ together with an Mai Tai DeepSee) featuring an ultra‐fast resonance scanner and sensitive hybrid detectors, we can image up to five fluorophores in
intravital
settings. This approach enables us to visualize lymphatics and blood vessels together with myeloid cells and lymphocytes in vivo examining changes in flow dynamics and cellular interaction (Fig 1).
For decades blood vasculature has been studied extensively, but our understanding of lymphatic functions remains poor. Lymphatics interact systemically and locally with the blood vasculature (Fig 2) and using these technique we have recently shown that dendritic cells are an important regulator in lymphatic function (Ivanov
et. al.
J Clin Invest. 2016 Apr 1;126(4):1581–91.). Many immune‐vasculature interactions can only be proven in the living animal, showing the interactions and impact of immune cells on vascular flow dynamics and vice versa. Our most recent
intravital
imaging studies allow the visualization of arterial resident macrophage dynamics within atherosclerotic plaque. Additionally, we are utilizing our intravital vascular imaging approaches to address the challenge of determining the role of distinct subsets of myeloid cells in glomerular disease and in murine tumor models (Fig 3). Furthermore, we are hoping these studies help to identify functional interactions of the vasculature and different immune cell populations as therapeutically targets for inflammatory diseases and for the enhancement of cancer immune therapy.
Support or Funding Information
BHZ was awarded a Scientist Development Grant (16SDG30480008) from the American Heart Association (AHA) 07/01/16‐06/30/19
Patients suffering from glioblastoma multiforme (GBM) face a poor prognosis with median survival of about 14 months. High recurrence rate and failure of conventional treatments are attributed to the ...presence of GBM cells with stem-like properties (GSCs). Metabolite profiles of 42 GSC lines established from the tumor tissue of adult GBM patients were screened with (1) H NMR spectroscopy and compared with human neural progenitor cells from human adult olfactory bulb (OB-NPCs) and from the developing human brain (HNPCs). A first subset (n=12) of GSCs exhibited a dramatic accumulation of the metabolite α-aminoadipate (αAAD), product of the oxidation of α-aminoadipic semialdehyde catalyzed by the ALDH7A1 aldehyde dehydrogenase (ALDH) family in lysine catabolism. αAAD was low/not detectable in a second GSC subset (n=13) with the same neural metabolic profile as well as in a third GSC subset (n=17) characterized by intense lipid signals. Likewise, αAAD was not detected in the spectra of OB-NPCs or HNPCs. Inhibition of mitochondrial ATP synthase by oligomycin treatment revealed that the lysine degradative pathway leading to αAAD formation proceeds through saccharopine, as usually observed in developing brain. Survival curves indicated that high αAAD levels in GSCs significantly correlated with poor patient survival, similarly to prostate and non-small-cell-lung cancers, where activity of ALDH7A1 correlates with tumor aggressiveness.
Dendritic cells (DCs) are key players in immunity and tolerance. Some DCs express c‐kit, the receptor for stem cell factor (SCF), nevertheless c‐kit functional role and the regulation of its ...expression in DCs are incompletely defined. We recently demonstrated that autocrine SCF sustains a pro‐survival circuit, and that SCF increases phospho‐AKT in c‐kit+ mouse bone marrow‐derived DCs (BMdDCs).
Herein we observed that CpG and PolyI:C, two stimuli mimicking bacterial and viral nucleic acids respectively, strongly inhibited c‐kit expression by BMdDCs and spleen DCs in vitro and in vivo. Experiments in IFNARI−/− mice showed that IFN‐I pathway was required for c‐kit down‐regulation in cDC1s, but only partially supported it in cDC2s. Furthermore, CpG and PolyI:C strongly inhibited c‐kit mRNA expression. In agreement with the reduced c‐kit levels, SCF pro‐survival activity was impaired. Thus in the presence of exogenously provided SCF, either PolyI:C or CpG induced spleen DC death in 2 days, while at earlier times IL‐6 and IL‐12 production were slightly increased. In contrast, SCF improved survival of unstimulated spleen DCs expressing high c‐kit levels.
Our studies suggest that c‐kit down‐modulation is a previously neglected component of DC response to CpG and PolyI:C, regulating DC survival and ultimately tuning immune response.
c‐kit down‐modulation is a previously neglected component of DC response to CpG and PolyI:C, regulating DC survival and ultimately tuning immune response.
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