Neoadjuvant radiotherapy in ER+, HER2+, and triple-negative -specific breast cancer based humanized tumor mice enhances anti-PD-L1 treatment efficacy.

Pre-operative radiation therapy is not currently integrated into the treatment protocols for breast cancer. However, transforming immunological "cold" breast cancers by neoadjuvant irradiation into their "hot" variants is supposed to elicit an endogenous tumor immune defense and, thus, enhance immunotherapy efficiency. We investigated cellular and immunological effects of sub-lethal, neoadjuvant irradiation of ER pos., HER2 pos., and triple-negative breast cancer subtypes in-vitro and in-vivo in humanized tumor mice (HTM). This mouse model is characterized by a human-like immune system and therefore facilitates detailed analysis of the mechanisms and efficiency of neoadjuvant, irradiation-induced "in-situ vaccination", especially in the context of concurrently applied checkpoint therapy. Similar to clinical appearances, we observed a gradually increased immunogenicity from the luminal over the HER2-pos. to the triple negative subtype in HTM indicated by an increasing immune cell infiltration into the tumor tissue. Anti-PD-L1 therapy divided the HER2-pos. and triple negative HTM groups into responder and non-responder, while the luminal HTMs were basically irresponsive. Irradiation alone was effective in the HER2-pos. and luminal subtype-specific HTM and was supportive for overcoming irresponsiveness to single anti-PD-L1 treatment. The treatment success correlated with a significantly increased T cell proportion and PD-1 expression in the spleen. In all subtype-specific HTM combination therapy proved most effective in diminishing tumor growth, enhancing the immune response, and converted non-responder into responder during anti-PD-L1 therapy. In HTM, neoadjuvant irradiation reinforced anti-PD-L1 checkpoint treatment of breast cancer in a subtype -specific manner. According to the "bench to bedside" principle, this study offers a vital foundation for clinical translating the use of neoadjuvant irradiation in the context of checkpoint therapy.

Subcellular localization of PD-L1 and cell-cycle-dependent expression of nuclear PD-L1 variants: implications for head and neck cancer cell functions and therapeutic efficacy.

The programmed cell death 1 ligand 1 (PD-L1)/programmed cell death protein 1 (PD-1) axis is primarily associated with immunosuppression in cytotoxic T lymphocytes (CTLs). However, mounting evidence is supporting the thesis that PD-L1 not only functions as a ligand but mediates additional cellular functions in tumor cells. Moreover, it has been demonstrated that PD-L1 is not exclusively localized at the cellular membrane. Subcellular fractionation revealed the presence of PD-L1 in various cellular compartments of six well-characterized head and neck cancer (HNC) cell lines, including the nucleus. Via Western blotting, we detected PD-L1 in its well-known glycosylated/deglycosylated state at 40-55 kDa. In addition, we detected previously unknown PD-L1 variants with a molecular weight at approximately 70 and > 150 kDa exclusively in nuclear protein fractions. These in vitro findings were confirmed with primary tumor samples from head and neck squamous cell carcinoma (HNSCC) patients. Furthermore, we demonstrated that nuclear PD-L1 variant expression is cell-cycle-dependent. Immunofluorescence staining of PD-L1 in different cell cycle phases of synchronized HNC cells supported these observations. Mechanisms of nuclear PD-L1 trafficking remain less understood; however, proximity ligation assays showed a cell-cycle-dependent interaction of the cytoskeletal protein vimentin with PD-L1, whereas vimentin could serve as a potential shuttle for nuclear PD-L1 transportation. Mass spectrometry after PD-L1 co-immunoprecipitation, followed by gene ontology analysis, indicated interaction of nuclear PD-L1 with proteins involved in DNA remodeling and messenger RNA (mRNA) splicing. Our results in HNC cells suggest a highly complex regulation of PD-L1 and multiple tumor cell-intrinsic functions, independent of immune regulation. These observations bear significant implications for the therapeutic efficacy of immune checkpoint inhibition.

Immune Checkpoint Profiling in Humanized Breast Cancer Mice Revealed Cell-Specific LAG-3/PD-1/TIM-3 Co-Expression and Elevated PD-1/TIM-3 Secretion.

Checkpoint blockade is particularly based on PD-1/PD-L1-inhibiting antibodies. However, an efficient immunological tumor defense can be blocked not only by PD-(L)1 but also by the presence of additional immune checkpoint molecules. Here, we investigated the co-expression of several immune checkpoint proteins and the soluble forms thereof (e.g., PD-1, TIM-3, LAG-3, PD-L1, PD-L2 and others) in humanized tumor mice (HTM) simultaneously harboring cell line-derived (JIMT-1, MDA-MB-231, MCF-7) or patient-derived breast cancer and a functional human immune system. We identified tumor-infiltrating T cells with a triple-positive PD-1, LAG-3 and TIM-3 phenotype. While PD-1 expression was increased in both the CD4 and CD8 T cells, TIM-3 was found to be upregulated particularly in the cytotoxic T cells in the MDA-MB-231-based HTM model. High levels of soluble TIM-3 and galectin-9 (a TIM-3 ligand) were detected in the serum. Surprisingly, soluble PD-L2, but only low levels of sPD-L1, were found in mice harboring PD-L1-positive tumors. Analysis of a dataset containing 3039 primary breast cancer samples on the R2 Genomics Analysis Platform revealed increased TIM-3, galectin-9 and LAG-3 expression, not only in triple-negative breast cancer but also in the HER2+ and hormone receptor-positive breast cancer subtypes. These data indicate that LAG-3 and TIM-3 represent additional key molecules within the breast cancer anti-immunity landscape.

Protein kinase C targeting of luminal (T-47D), luminal/HER2-positive (BT474), and triple negative (HCC1806) breast cancer cells in-vitro with AEB071 (Sotrastaurin) is efficient but mediated by subtype specific molecular effects.

PURPOSE: Protein kinase C (PKC) plays a pivotal role in malignant cell proliferation, apoptosis, invasiveness and migration. However, its exploitation as therapeutic target in breast cancer has been merely explored. Here were evaluated the AEB071 (Sotrastaurin™) treatment efficiency of breast cancer cell lines derived from estrogen receptor positive (T-47D), estrogen/HER2 receptor positive (BT474), and triple negative (HCC1806) breast cancer cells under 2D (monolayer) and 3D (multicellular tumor spheroids) culture conditions. Additionally, spheroid cocultures of BC and N1 fibroblasts were analyzed. METHODS: We quantitatively assessed the proliferation capacity of breast cancer cells and fibroblasts as a function of AEB071 treatment using flow cytometry. The activities of PKC isoforms, substrates, and key molecules of the PKC signaling known to be involved in the regulation of tumor cell proliferation and cellular survival were additionally evaluated. Moreover, a multigene expression analysis (PanCancer Pathways assay) using the nanoString™ technology was applied. RESULTS: All breast cancer cell lines subjected to this study were sensitive to AEB071 treatment, whereby cell proliferation in 2D culture was considerably (BT474) or moderately (HCC1806) retarded in G0/G1 or in G2/M phase (T-47D) of the cell cycle. Regardless of the breast cancer subtype the efficiency of AEB071 treatment was significantly lower in the presence of N1 fibroblast cells. Subtype specific driver molecules, namely IL19, c-myb, and NGFR were mostly affected by the AEB071 treatment. CONCLUSION: A combined targeting of PKC and a subtype specific driver molecule might complement specified breast cancer treatment.

Differential Expression of PD-L1 during Cell Cycle Progression of Head and Neck Squamous Cell Carcinoma.

The expression of PD-L1 by tumor cells is mainly associated with its immunosuppressive effect. In fact, PD-1/PD-L1 immune checkpoint inhibitors demonstrated remarkable effects in advanced cancer patients including HNSCC. In this context, irradiation is currently being investigated as a synergistic treatment modality to immunotherapy. However, the majority of HNSCC patients still show little improvement or even hyperprogression. Interestingly, there is increasing evidence for additional cell-intrinsic functions of PD-L1 in tumor cells. In previous studies, we showed that PD-L1 has a strong influence on proliferation, migration, invasion, and survival after irradiation. We demonstrated that cellular expression and localization of PD-L1 differed depending on sensitivity to irradiation. Here, we show that PD-L1 is also differentially expressed during cell cycle progression of HNSCC. Furthermore, cellular localization of PD-L1 also changes depending on a particular cell cycle phase. Moreover, distinct observations occurred depending on the general differentiation status. Overall, the function of PD-L1 cannot be generalized. Rather, it depends on the differentiation status and microenvironment. PD-L1 expression and localization are variable, depending on different factors. These findings may provide insight into why differential response to PD-1/PD-L1 antibody therapy can occur. Detailed understanding of cell-intrinsic PD-L1 functions will further allow antibody-based immunotherapy to be optimized.

The Chemokine CX3CL1 Improves Trastuzumab Efficacy in HER2 Low-Expressing Cancer In Vitro and In Vivo.

A crucial mode of action of trastuzumab is the labeling of HER2-positive (HER2+) tumor cells for the eradication by natural killer (NK) cells, a process called antibody-dependent cellular cytotoxicity (ADCC). However, despite widespread HER2 expression among cancer entities, only a fraction, with robust HER2 overexpression, benefits from trastuzumab therapy. ADCC requires both sufficient lymphocytic infiltration and close binding of the immune cells to the antibody-tagged tumor cells. We hypothesized that the chemokine CX3CL1 could improve both processes, as it is synthesized as a membrane-bound, adhesive form that is eventually cleaved into a soluble, chemotactic protein. Here, we show that CX3CL1 overexpression is a positive prognostic marker in breast cancer. CX3CL1 overexpression attracted tumor-suppressive lymphocytes, including NK cells, and inhibited tumor growth and lung metastasis in the syngeneic 4T1 breast cancer mouse model. In HER2+ SKBR3, MDA-MB-453, and HT-29 tumor cells, CX3CL1 overexpression increased NK cell-mediated cytotoxicity in vitro and acted synergistically with trastuzumab. Even though CX3CL1 did not further improve trastuzumab efficacy in vivo in the trastuzumab-sensitive MDA-MB-453 model, it compensated for NK-cell depletion and prolonged survival. In the HER2 low-expressing HT-29 model, however, CX3CL1 overexpression not only prolonged survival time but also overcame trastuzumab resistance in a partly NK cell-dependent manner. Taken together, these findings identify CX3CL1 as a feasible pharmacologic target to enable trastuzumab therapy in HER2 low-expressing cancers and render it a potential predictive biomarker to determine therapy responders.

In vivo detection of breast cancer liver metastases in humanized tumour mice using tumour specific contrast agent BR55®.

OBJECTIVE: To investigate the diagnostic accuracy of high-resolution ultrasound (HRU) for the detection of hepatic metastases of breast cancer in a humanized tumour mouse (HTM) using clinical standard technology. In addition, the efficiency of standard contrast-enhanced ultrasound (CEUS) [microbubbles of sulphur hexafluoride] and CEUS using a novel VEGFR2-targeted contrast agent [BR55®] was examined. METHODS: A total of 14 HTM were sonographically examined twice. In addition to a human immune system, the animals developed hepatic tumour lesions after intrahepatic injection of BT-474 breast cancer cells. Digital cine loops from the arterial phase (15-35 sec), the portal venous phase (35-90 sec) and the late phase (3-15 min) of the entire liver were analysed. Data were correlated to histopathology. RESULTS: After 9 months, half of the mice (7/14) revealed the development of hepatic breast cancer metastases. The detection limit was 1 mm tumour diameter. In particular, the use of targeted contrast media reduced the needed tumour diameter and helped to precisely classify tumour tissue. In 93% (13/14), the findings of ultrasound could be approved by histological examination by the pathologist. CONCLUSIONS: This study in HTM demonstrated the high feasibility of tumour specific contrast media and standard HRU contrast agents to detect early liver metastases.

Humanized mouse model: Hematopoietic stemcell transplantation and tracking using short tandem repeat technology.

INTRODUCTION: Models of mice carrying a human immune system, so-called humanized mice, are used increasingly as preclinical models to bridge the gap between model organisms and human beings. Challenges of the humanized mouse model include finding suitable sources for human hematopoietic stem cells (HSC) and reaching sufficient engraftment of these cells in immunocompromised mice. METHODS: In this study, we compared the use of CD34+ HSC from cord blood (CB) vs HSC from adult mobilized peripheral blood. Furthermore, we developed a simple and highly specific test for donor identification in humanized mice by applying the detection method of short tandem repeats (STR). RESULTS: It was found that, in vitro, CB-derived and adult HSC show comparable purity, viability, and differentiation potential in colony-forming unit assays. However, in vivo, CB-derived HSC engrafted to a significantly higher extent in NOD.Cg-Prkdcscid IL2rγtm1Wjl /SzJ (NSG) mice than adult HSC. Increasing the cell dose of adult HSC or using fresh cells without cryopreservation did not improve the engraftment rate. Interestingly, when using adult HSC, the percentage of human cells in the bone marrow was significantly higher than that in the peripheral blood. Using the STR-based test, we were able to identify and distinguish human cells from different donors in humanized mice and in a humanized allogeneic transplantation model. CONCLUSION: From these findings, we conclude that adult mobilized HSC are less suitable for generating a humanized immune system in mice than CB-derived cells.

TIGIT+ iTregs elicited by human regulatory macrophages control T cell immunity.

Human regulatory macrophages (Mreg) have shown early clinical promise as a cell-based adjunct immunosuppressive therapy in solid organ transplantation. It is hypothesised that recipient CD4+ T cell responses are actively regulated through direct allorecognition of donor-derived Mregs. Here we show that human Mregs convert allogeneic CD4+ T cells to IL-10-producing, TIGIT+ FoxP3+-induced regulatory T cells that non-specifically suppress bystander T cells and inhibit dendritic cell maturation. Differentiation of Mreg-induced Tregs relies on multiple non-redundant mechanisms that are not exclusive to interaction of Mregs and T cells, including signals mediated by indoleamine 2,3-dioxygenase, TGF-ß, retinoic acid, Notch and progestagen-associated endometrial protein. Preoperative administration of donor-derived Mregs to living-donor kidney transplant recipients results in an acute increase in circulating TIGIT+ Tregs. These results suggest a feed-forward mechanism by which Mreg treatment promotes allograft acceptance through rapid induction of direct-pathway Tregs.

Single- and two-photon imaging of human micrometastases and disseminated tumour cells with conjugates of nanobodies and quantum dots.

Early detection of malignant tumours and, especially, micrometastases and disseminated tumour cells is still a challenge. In order to implement highly sensitive diagnostic tools we demonstrate the use of nanoprobes engineered from nanobodies (single-domain antibodies, sdAbs) and fluorescent quantum dots (QDs) for single- and two-photon detection and imaging of human micrometastases and disseminated tumour cells in ex vivo biological samples of breast and pancreatic metastatic tumour mouse models expressing human epidermal growth factor receptor 2 (HER2) or carcinoembryonic antigen (CEA). By staining thin (5-10 µm) paraffin and thick (50 µm) agarose tissue sections, we detected HER2- and CEA-positive human tumour cells infiltrating the surrounding tissues or metastasizing to different organs, including the brain, testis, lung, liver, and lymph nodes. Compared to conventional fluorescently labelled antibodies the sdAb-HER2-QD and sdAb-CEA-QD nanoprobes are superior in detecting micrometastases in tissue sections by lower photobleaching and higher brightness of fluorescence signals ensuring much better discrimination of positive signals versus background. Very high two-photon absorption cross-sections of QDs and small size of the nanoprobes ensure efficient imaging of thick tissue sections unattainable with conventional fluorescent probes. The nanobody-QD probes will help to improve early cancer diagnosis and prognosis of progression by assessing metastasis.

Regulation of Programmed Death Ligand 1 (PD-L1) Expression in Breast Cancer Cell Lines In Vitro and in Immunodeficient and Humanized Tumor Mice.

Programmed death ligand 1 (PD-L1) expression is an efficient strategy of tumor cells to escape immunological eradiation. However, only little is known about the factors that affect the cellular expression levels. Here we assessed the PD-L1 expression on different breast cancer cell lines under standard in vitro culture conditions and as a function of Epirubicin or Paclitaxel treatment. Moreover, we evaluated the expression in immunodeficient tumor mice as well as in humanized tumor mice (i.e., in the presence of a human immune system). We found highest PD-L1 levels in JIMT-1 and MDA-MB-231 cells. Epirubicin treatment caused a decrease and Paclitaxel treatment an increased PD-L1 expression in MDA-MB-231 cells. In addition, we identified nuclear PD-L1 in MDA-MB-231 cells. All in vivo transplanted breast cancer cell lines downregulated PD-L1 expression compared to their in vitro counterpart. Neither the gene copy number nor the presence of human immune system in humanized tumor mice had an effect on the PD-L1 content. We demonstrate that the degree of PD-L1 expression amongst breast cancer cell lines varies considerably. In addition, cytotoxic treatments and other extrinsic parameters differentially affect the expression. Hence, further investigations including in vivo evaluations are necessary to understand PD-L1 regulation for advanced breast cancer stratification.

Humanized Mice with Subcutaneous Human Solid Tumors for Immune Response Analysis of Vaccinia Virus-Mediated Oncolysis.

Oncolytic vaccinia virus (VACV) therapy is an alternative cancer treatment modality that mediates targeted tumor destruction through a tumor-selective replication and an induction of anti-tumor immunity. We developed a humanized tumor mouse model with subcutaneous human tumors to analyze the interactions of VACV with the developing tumors and human immune system. A successful systemic reconstitution with human immune cells including functional T cells as well as development of tumors infiltrated with human T and natural killer (NK) cells was observed. We also demonstrated successful in vivo colonization of such tumors with systemically administered VACVs. Further, a new recombinant GLV-1h376 VACV encoding for a secreted human CTLA4-blocking single-chain antibody (CTLA4 scAb) was tested. Surprisingly, although proving CTLA4 scAb's in vitro binding ability and functionality in cell culture, beside the significant increase of CD56bright NK cell subset, GLV-1h376 was not able to increase cytotoxic T or overall NK cell levels at the tumor site. Importantly, the virus-encoded ß-glucuronidase as a measure of viral titer and CTLA4 scAb amount was demonstrated. Therefore, studies in our "patient-like" humanized tumor mouse model allow the exploration of newly designed therapy strategies considering the complex relationships between the developing tumor, the oncolytic virus, and the human immune system.

IL-15 enhances the anti-tumor activity of trastuzumab against breast cancer cells but causes fatal side effects in humanized tumor mice (HTM).

Cancer immunotherapy has been shown to enhance established treatment regimens. We evaluated the potential reinforcing effect of IL-15 in trastuzumab treated humanized tumor mice (HTM) which were generated by concurrent transplantation of neonatal NOD-scid IL2Rγnull mice with human hematopoietic stem cells (HSC) and HER2 positive breast cancer cells (metastasizing SK-BR-3, solid tumor forming BT474).We found that trastuzumab treatment efficacy mainly depends on the immediate anti-tumorigenic cellular effect which is significantly enhanced by tumor interacting immune cells upon cotransplantion of HSC. However, trastuzumab treatment caused elevated CD44 expression on tumor cells that metastasized into the lung and liver but did not hinder tumor cell dissemination into the bone marrow. Moreover, in a number of SK-BR-3-transplanted animals disseminated CD44high/CD24low tumor cells lost trastuzumab sensitivity. Concerning the FcγRIIIa polymorphism, trastuzumab treatment efficiency in HTM was higher in mice with NK-cells harboring the high affinity FcγRIIIa compared to those with low affinity FcγRIIIa. In contrast, IL-15 caused the strongest NK-cell activation in heterozygous low affinity FcγRIIIa animals. Although IL-15 enhanced the trastuzumab mediated tumor defense, an unspecific immune stimulation resulted in preterm animal death due to systemic inflammation. Overall, treatment studies based on "patient-like" HTM revealed critical and adverse immune-related mechanisms which must be managed prior to clinical testing.

IRAK-M Expression in Tumor Cells Supports Colorectal Cancer Progression through Reduction of Antimicrobial Defense and Stabilization of STAT3.

Colorectal cancer (CRC) is associated with loss of epithelial barrier integrity, which facilitates the interaction of the immunological microenvironment with the luminal microbiome, eliciting tumor-supportive inflammation. An important regulator of intestinal inflammatory responses is IRAK-M, a negative regulator of TLR signaling. Here we investigate the compartment-specific impact of IRAK-M on colorectal carcinogenesis using a mouse model. We demonstrate that IRAK-M is expressed in tumor cells due to combined TLR and Wnt activation. Tumor cell-intrinsic IRAK-M is responsible for regulation of microbial colonization of tumors and STAT3 protein stability in tumor cells, leading to tumor cell proliferation. IRAK-M expression in human CRCs is associated with poor prognosis. These results suggest that IRAK-M may be a potential therapeutic target for CRC treatment.

An Emerging Approach for Parallel Quantification of Intracellular Protozoan Parasites and Host Cell Characterization Using TissueFAXS Cytometry.

Characterization of host-pathogen interactions is a fundamental approach in microbiological and immunological oriented disciplines. It is commonly accepted that host cells start to change their phenotype after engulfing pathogens. Techniques such as real time PCR or ELISA were used to characterize the genes encoding proteins that are associated either with pathogen elimination or immune escape mechanisms. Most of such studies were performed in vitro using primary host cells or cell lines. Consequently, the data generated with such approaches reflect the global RNA expression or protein amount recovered from all cells in culture. This is justified when all host cells harbor an equal amount of pathogens under experimental conditions. However, the uptake of pathogens by phagocytic cells is not synchronized. Consequently, there are host cells incorporating different amounts of pathogens that might result in distinct pathogen-induced protein biosynthesis. Therefore, we established a technique able to detect and quantify the number of pathogens in the corresponding host cells using immunofluorescence-based high throughput analysis. Paired with multicolor staining of molecules of interest it is now possible to analyze the infection profile of host cell populations and the corresponding phenotype of the host cells as a result of parasite load.

Topotecan-induced ABCG2 expression in MCF-7 cells is associated with decreased CD24 and EpCAM expression and a loss of tumorigenicity.

Human breast cancer shows a considerable heterogeneity regarding the expression of CD24, CD44, EpCAM, and HER2. These markers are involved in cell adhesion, migration, and proliferation, and thus affect metastasis and, in turn, patient's outcome. The ATP-driven efflux pump (ABC transporter) breast cancer resistance protein (BCRP, ABCG2) is known to confer resistance to a wide variety of structurally unrelated cytostatics and defines subpopulations with enhanced tumor-initiating capacity. The expression of ABCG2 can be induced by treatment with different cytostatic drugs. Concurrent effects of such treatments on the expression of the aforementioned marker proteins and cellular properties related to cancer-initiating cells have not been examined thoroughly. Here, we investigated the effect of the ABCG2 substrate topotecan on the MCF-7 breast cancer cell line and analyzed CD24, CD44, EpCAM, and HER2 expression by flow cytometry. Moreover, we examined the impact of topotecan treatment on the sphere-forming ability in vitro and the tumorigenicity in immunodeficient NMRI-nu/nu and NSG mice. We found an elevated ABCG2 expression in MCF-7 cells in the presence of 500 nM topotecan. Compared with untreated MCF-7 cells, the application of topotecan induced a subpopulation with decreased CD24/EpCAM expression, whereas CD44 expression remained largely unchanged. Topotecan-treated cells showed an impaired mammosphere formation capacity in vitro and reduced tumorigenicity in immunodeficient mice. The data indicate that ABCG2 induction is not necessarily linked to increased tumorigenicity and suggest a major role of CD24 and EpCAM for the preservation of self-renewal capacity in MCF-7 cells and tumor outgrowth in vivo.

RORγt(+) hematopoietic cells are necessary for tumor cell proliferation during colitis-associated tumorigenesis in mice.

Colorectal cancer (CRC) is one of the most common tumor entities. In patients with inflammatory bowel diseases, the development of colitis-associated colon cancer is considered a dangerous long-term complication. IL-17A and the transcription factor retinoic acid receptor-related orphan receptor γt (RORγt) play fundamental roles in the pathogenesis of inflammatory bowel diseases; in human studies, we detected a dense infiltration of RORγt-dependent CD4(+) IL17A(+) T helper (Th)17 cells in specimens of CRC, ulcerative colitis, and ulcerative colitis-associated colorectal cancer. However, the mechanistic role of RORγt(+) hematopoietic cells in colitis-associated tumorigenesis remains unclear. To investigate colitis-associated colon tumorigenesis, we conducted studies in the AOM+DSS mouse model that revealed the importance of RORγt for colon tumor progression. In the absence of RORγt-dependent Th17 lymphocytes, mice showed signs of intense chronic colitis, but developed significantly fewer macroscopic tumor nodules. The reduction of tumor development in RORγt(-/-) mice was not due to reduced colon tumor initiation. However, the proliferation rate of tumor cells was reduced in the absence of RORγt-dependent Th17 cells and tumor cells showed pronounced signs of senescence-associated epigenetic and lysosomal changes. These results indicate an important role for the immunological milieu in colitis-associated cancer, which is shaped in-part by RORγt-dependent Th17 lymphocytes that support CRC growth.

Staphylococcus aureus Infection in Humanized Mice: A New Model to Study Pathogenicity Associated With Human Immune Response.

BACKGROUND: Staphylococcus aureus is a common pathogen among humans worldwide, with an increasing prevalence of multidrug resistance. The understanding of virulence factors inducing pathogenicity is still incomplete, and thus far the transfer of results from animal studies into successful clinical trials has been difficult. METHODS: In this study, we established an S. aureus infection model in mice engrafted with a human immune system, compared it with infected wild-type and nonhumanized mice, and investigated pathogenesis in these models. RESULTS: Staphylococcus aureus infection was aggravated in humanized mice, compared with wild-type or nonengrafted mice. The humanized mice displayed a significantly reduced survival percentage, increased weight loss, and a more-rapid increase in bacterial burden. In addition, S. aureus infection induced T-cell activation, apoptosis, and Fas receptor expression in humanized but not wild-type mice. CONCLUSIONS: Our findings demonstrate the different pathogenetic mechanisms in wild-type and humanized mice and the possible benefit of including humanized mice in future studies involving S. aureus as a prior step to human clinical trials.

Laser ablation-inductively coupled plasma mass spectrometry: an emerging technology for detecting rare cells in tissue sections.

Administering immunoregulatory cells to patients as medicinal agents is a potentially revolutionary approach to the treatment of immunologically mediated diseases. Presently, there are no satisfactory, clinically applicable methods of tracking human cells in patients with adequate spatial resolution and target cell specificity over a sufficient period of time. Laser ablation-inductively coupled plasma mass spectrometry (LA-ICP-MS) represents a potential solution to the problem of detecting very rare cells in tissues. In this article, this exquisitely sensitive technique is applied to the tracking of gold-labeled human regulatory macrophages (Mregs) in immunodeficient mice. Optimal conditions for labeling Mregs with 50-nm gold particles were investigated by exposing Mregs in culture to variable concentrations of label: Mregs incubated with 3.5 × 10(9) particles/ml for 1 h incorporated an average of 3.39 × 10(8) Au atoms/cell without loss of cell viability. Analysis of single, gold-labeled Mregs by LA-ICP-MS registered an average of 1.9 × 10(5) counts/cell. Under these conditions, 100% labeling efficiency was achieved, and label was retained by Mregs for ≥36 h. Gold-labeled Mregs adhered to glass surfaces; after 24 h of culture, it was possible to colabel these cells with human-specific (154)Sm-tagged anti-HLA-DR or (174)Yb-tagged anti-CD45 mAbs. Following injection into immunodeficient mice, signals from gold-labeled human Mregs could be detected in mouse lung, liver, and spleen for at least 7 d by solution-based inductively coupled plasma mass spectrometry and LA-ICP-MS. These promising results indicate that LA-ICP-MS tissue imaging has great potential as an analytical technique in immunology.

Co-transplantation of human hematopoietic stem cells and human breast cancer cells in NSG mice: a novel approach to generate tumor cell specific human antibodies.

Humanized tumor mice (HTM) were generated by the co-transplantation of human hematopoietic stem cells and human breast cancer cells overexpressing HER2 into neonatal NOD-scid IL2Rγ(null) (NSG) mice. These mice are characterized by the development of a human immune system in combination with human breast cancer growth. Due to concurrent transplantation into newborn mice, transfer of MHC-mismatched tumor cells resulted in solid coexistence and immune cell activation (CD4(+) T cells, natural killer cells, and myeloid cells), but without evidence for rejection. Histological staining of the spleen of HTM revealed co-localization of human antigen-presenting cells together with human T and B cells allowing MHC-dependent interaction, and thereby the generation of T cell-dependent antibody production. Here, we investigated the capability of these mice to generate human tumor-specific antibodies and correlated immunoglobulin titers with tumor outgrowth. We found detectable IgM and also IgG amounts in the serum of HTM, which apparently controlled tumor development when IgG serum concentrations were above 10 µg/ml. Western blot analyses revealed that the tumor-specific antibodies generated in HTM did not recognize HER2/neu antigens, but different, possibly relevant antigens for breast cancer therapy. In conclusion, HTM offer a novel approach to generate complete human monoclonal antibodies that do not require further genetic manipulation (e. g., humanization) for a potential application in humans. In addition, efficacy and safety of the generated antibodies can be tested in the same mouse model under human-like conditions. This might be of particular interest for cancer subtypes with no currently available antibody therapy.